Dyeing method for automated system

Abstract

Staining devices and methods for automated systems are provided herein. A dyeing method includes disposing a substrate proximate a dyed surface to establish an overlap between the dyed surface and the substrate. The staining method further includes maintaining a gap between the stained surface and the substrate to define a chromosome product according to the overlap and the gap, where the chromosome product has one or more meniscus edges. The staining method further includes disposing a portion of the staining fluid over the staining surface and distributing the staining fluid over the substrate.

Description

Technical Field

[0001] This invention relates to systems, methods, and apparatuses configured to facilitate sample handling, including automated sample handling and rapid on-site sample evaluation. Background Technology

[0002] Rapid On-Site Evaluation (ROSE) is an important aspect of biopsy procedures, reducing the number of needle passes, improving patient safety, and enhancing diagnostic yield. The purpose of ROSE is to determine whether the biopsy sample contains sufficient cells and tissue for a definitive diagnosis when analyzed in a pathology laboratory. Other uses of ROSE include informing tissue collection and triage, and providing preliminary diagnosis to interventional specialists.

[0003] Traditionally, in ROSE, a cytologist or cytopathologist smears or touch-presses the biopsy sample onto a glass slide to create a thin layer of cellular material. The slide is then manually stained with rapid staining agents such as Diff Quik, toluidine blue, or others to increase contrast between the different biological elements. Finally, the slide is examined under an optical microscope by a cytologist for adequacy, triage, and / or preliminary diagnosis.

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

[0005] However, due to a shortage of cytologists, logistical challenges, and the limited resources of pathologists, ROSE is used for only about half of non-dermatological biopsy procedures. These challenges are amplified in procedures such as bronchoscopic lung biopsy, endobronchial ultrasound (EBUS) lymph node staging, and percutaneous CT-guided biopsy, as the long procedural times place additional strain on the cytology department's resources. Furthermore, ROSE adequacy and diagnostic accuracy are also affected by operator variability in both slide preparation and interpretation—no two cytologists will prepare the same slides or make the same adequacy call—which is often a source of frustration and time delays for interventional staff. For complex procedures such as bronchoscopic lung biopsy, pathology labs cannot provide a definitive diagnosis in 20–40% or more of biopsies—requiring repeat biopsy procedures—even using conventional ROSE methods.

[0006] Given the limitations of traditional ROSE, it is not surprising to explore alternative methods to improve adequacy assessment and on-site diagnostic procedures. Several devices exist that provide digitization and transport of ROSE slides, or remote robotic microscopes that enable cytopathologists to view ROSE slides remotely. However, such devices still rely on operating room cytologists to prepare the slides, which does not overcome the logistical and operator variability issues associated with certain ROSE procedures.

[0007] Several potential solutions are being explored, such as Raman scattering microscopy for tissue resection and needle-based confocal laser microscopy. While the principles are appealing, these methods do not match the resolution and fidelity of imaging tissue on a glass slide. Furthermore, these methods create unfamiliar images, requiring new training for interventional specialists and pathologists.

[0008] Therefore, improved sample processing methods and techniques are needed to reduce sample processing time, improve the uniformity of sample processing, and reduce the operator time required for sample processing. Summary of the Invention

[0009] In one embodiment, a method for distributing a staining solution on a substrate is provided. The method includes arranging the substrate near a staining surface to create an overlap between the staining surface and the substrate; maintaining a gap between the staining surface and the substrate to define a chromosome volume / staining volume based on the overlap and the gap, the chromosome volume having one or more meniscus edges; arranging a portion of the staining solution on the staining surface; and distributing the staining solution on the substrate. Attached Figure Description

[0010] The accompanying drawings, incorporated herein and forming part of this specification, illustrate various embodiments of systems, methods, and apparatuses for rapid on-site cytological assessment. The drawings, together with this description, further illustrate the principles and enable those skilled in the art to make and use the methods, systems, and apparatuses described herein. These drawings are provided to illustrate various features of the embodiments described herein, and are not necessarily drawn to scale. In these drawings, the same reference numerals indicate the same or functionally similar elements.

[0011] Figure 1 A system for performing Rapid Field Evaluation (ROSE) to perform sample analysis is shown.

[0012] Figure 2A A method for distributing dye solution on a substrate, consistent with its embodiments, is shown.

[0013] Figure 2B and Figure 2C A schematic diagram of a method for distributing staining solution consistent with its embodiments is provided.

[0014] Figures 3A-3K The various stages of the process of distributing the dye solution onto the substrate, consistent with its embodiments, are shown.

[0015] Figures 4A-4J The various stages of the process of distributing the dye solution onto the substrate, consistent with its embodiments, are shown.

[0016] Figure 5A Figure 5V illustrates the stages of the process of distributing the dye solution onto the substrate, consistent with its embodiment.

[0017] Figure 6A Figure 6N illustrates the stages of the process of distributing the dye solution onto the substrate, consistent with its embodiment.

[0018] Figures 7A-7D The various stages of the process of distributing the dye solution onto the substrate, consistent with its embodiments, are shown.

[0019] Figures 8A-8B A staining cartridge, including a staining reservoir, is shown, consistent with its embodiments.

[0020] Figure 9 The staining structure consistent with its embodiment is shown. Detailed Implementation

[0021] The following detailed description is merely exemplary in nature and is not intended to limit the invention or its application and uses. Although the description of the invention is set out in the context of systems, methods, and apparatuses for facilitating automated ROSE techniques, this disclosure should not be considered so limiting. For example, while systems, methods, and apparatuses may be discussed herein with respect to ROSE of biopsy samples, any biological sample may be suitable for analysis by means of its embodiments. Modifications may be made to the various embodiments described herein without departing from the spirit and scope of the invention. Therefore, the following detailed description is not intended to be restrictive. Furthermore, there is no intention to be bound by any express or implied theory set forth in the foregoing technical fields, background art, summary of the invention, or the following detailed description.

[0022] This disclosure addresses the problems outlined above. It relates to a robotic automated staining and sample processing apparatus that rapidly generates consistent, high-resolution cytological images from raw biopsy samples collected by interventional personnel, and is therefore suitable for ROSE. The images can be displayed directly to the interventional specialist on the machine or transmitted to a cytopathologist for remote consultation. Artificial intelligence algorithms can assist the cytopathologist by identifying important regions of interest and cell types. Through highly reproducible sample preparation and imaging, interventional specialists can directly obtain fully automated adequacy and diagnosis (using artificial intelligence) without the need for remote consultation. Using the methods described herein, interventional specialists and remote cytopathologists can view high-resolution smear images prepared and stained in a familiar manner without the need for on-site cytologists. Therefore, the various embodiments of the disclosure provided herein can be used to reduce operator time for ROSE, increase throughput, reduce time to obtain results, and increase the consistency of results.

[0023] The embodiments disclosed herein facilitate rapid staining and evaluation of samples using sample separation techniques that utilize minimal or reduced sample amounts compared to other methods. In each embodiment, the systems, methods, and apparatus described herein can be used in conjunction with other systems, methods, and apparatuses to evaluate, measure, or assess the therapeutic effect on sample tissue. For example, after the delivery of treatment (e.g., drug, ablation, etc.), tissue can be collected and stained to determine the efficacy of the treatment. For instance, tissue sampled from a tumor periphery after ablation can be stained according to the systems, methods, and apparatus described herein and evaluated to ensure or determine that the region has been adequately ablated at the cellular level. Similarly, tissue sampled from a tumor periphery after intratumoral drug injection can be stained according to the embodiments described herein and evaluated to ensure or determine that the drug has adequately penetrated the tissue at the cellular level.

[0024] Automated sample staining offers significant benefits compared to traditional manual staining techniques, particularly in providing faster staining and more economical use of staining fluids. Samples can be stained more quickly, and less staining fluid is wasted. Furthermore, when used as part of an automated processing line, automated sample staining eliminates the need to interrupt the line to allow for manual intervention. The automated sample staining techniques discussed in this paper utilize fluid properties and behaviors, including surface tension, adhesion, capillary action, and wicking. The utilization of these properties and behaviors can be facilitated by employing automated staining techniques such as those discussed herein—which minimize bubble formation while eliminating any bubbles that do form. Bubble formation can be particularly detrimental to automated systems, as any interruption in the fluid can reduce or prevent effects such as wicking, capillary action, surface tension, adhesion, and wicking. Furthermore, staining techniques such as those discussed herein can enhance and / or optimize the fluid properties and behaviors discussed above.

[0025] This disclosure describes apparatus, systems, and methods for facilitating staining and sample processing. The sample staining methods described herein involve the arrangement of staining fluids and the staining of samples (e.g., tissue samples or other biological samples). The sample staining apparatuses and methods described herein facilitate rapid, reproducible, and automated sample staining with minimal operator intervention. Each of the described methods and apparatuses is compatible with a variety or number of actuators and systems configured to facilitate the performance of the described methods using the disclosed apparatus structures. Combinations of the presented features are discussed by way of example only, and each of the sample staining methods, apparatuses, and systems described herein can be used or utilized with alternative methods, systems, and apparatuses as appropriate. Furthermore, individual aspects of each of the methods, systems, and apparatuses described herein can be used alone or in any suitable combination with any other individual aspect of the methods, systems, and apparatuses described herein. Some of these individual combinations may be discussed herein for illustrative or exemplary purposes. Although the wide variety of all such combinations precludes a separate description of each combination herein, it should be understood that all such combinations fall within the scope of this disclosure.

[0026] For example, any method or part of a method described herein can be implemented with appropriate portions of the apparatus and systems described herein without requiring all of the apparatus and systems described herein, and / or can be used with alternative apparatus and systems. Some methods or parts of a method can also be implemented manually with appropriate portions of the apparatus described herein and / or with the systems described herein using different apparatus. The apparatus described herein can be implemented with appropriate portions of the methods and systems described herein without requiring all of the methods and systems described herein, and / or can be used with alternative methods and systems. The systems described herein can be implemented with appropriate portions of the methods and apparatus described herein without requiring all of the methods and apparatus described herein, and / or can be used with alternative methods and apparatus.

[0027] In various embodiments, the integrated sample staining and processing system 100 can be configured to work with a common glass slide (or other suitable substrate) serving as a sample substrate. This setup may impose certain requirements on the precision manipulators, grippers, and other positioning actuators of the integrated sample staining and processing system 100. In various embodiments, as described herein, the sample staining and processing system 100 may include staining structures configured to facilitate the staining methods described herein. The staining structures described herein may be permanent or disposable, and may be integral or removable relative to the sample staining and processing system. Various embodiments of staining structures include, for example, staining cartridges 303, as described below. Various features of the staining cartridge 303 as described herein can be included in permanent or disposable, as well as integral or removable staining structures. The sample staining and processing system 100 can be configured to receive or include one or more specially constructed cartridges or staining structures, such as the staining cartridge 303 described below. Such a staining cartridge 303 may include some or all of the sample staining components required for a particular sample staining sequence. Sample staining components may include, but are not limited to, staining plates or multiple staining plates, sample slides or multiple sample slides, one or more staining solutions, one or more fixatives, one or more washing solutions, and one or more staining solution or other liquid storage units. As used herein, “staining solution” may refer to liquid-based staining agents (e.g., combinations of immunohistochemical (IHC) staining agents, fluorescent staining agents, antibody staining agents, biological staining agents, any staining agents for selected biological entities such as genes or proteins, and / or any other staining agents or staining solutions known in the art) and / or any other liquids used in the staining process, such as fixatives and washing solutions (e.g., water, alcohol, etc.). Staining cassettes may hold or guide each element in a sufficiently relative position as required by the sample staining steps. By incorporating appropriate precision into the staining cassettes, the precision requirements of the processing system can be correspondingly reduced. Furthermore, staining cassettes may also be configured to accommodate all contaminated elements for safe disposal and to prevent contamination of the processing system.

[0028] In a further embodiment, the integrated sample staining and processing system 100 can be configured to work with a standard glass slide (or other suitable substrate) serving as a sample substrate without the use of a cassette. In such an embodiment, the sample substrate can be handled, manipulated, controlled, etc., using actuators of the system, including manipulators, grippers, rollers, clamps, and any other suitable actuators.

[0029] This 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, “top” of a structure or apparatus may refer to the portion of the structure or apparatus facing upwards during use as described herein. Relative directional terms used herein are not limiting and do not restrict the orientation, position, angle, or function of the structures and apparatus discussed herein. Furthermore, the methods, systems, and apparatuses discussed herein are not limited to use in the orientations described herein. For example, although a dyeing box is described as having a dyeing plate sliding “above” the dyeing box, this disclosure is not limited to this arrangement. One or more aspects / orientations of a system may be reversed relative to the orientations disclosed herein without departing from the scope of this disclosure.

[0030] Figure 1 A rapid field assessment system consistent with its embodiments is illustrated. The rapid field assessment system 100 may include various components. These components may be housed on a mobile cart 101 or as a benchtop system. Components may include a wheeled cart chassis; an integrated sample staining and processing system 100 for preparing, staining, and imaging biopsy samples; a display 102 for viewing the prepared and imaged samples; and a user interface 103 including one or more user input devices; a storage for disposable cartridges (in which biopsy samples may be loaded); and one or more consumable repositories. A waste fluid tank may also be included. The integrated sample staining and processing system 100 may include one or more input slots in which disposable cartridges containing samples may be loaded; one or more output slots for retrieving stained slides for future analysis; and an area for retrieving used cartridges to be discarded. Additionally, the trolley may also contain controls (such as one or more user input devices for user interface 103) to facilitate input of system parameters and execution of operations (such as manipulating microscope images on the screen by panning, zooming, and jumping to various areas of interest).

[0031] The integrated sample staining and processing system 100 may include the necessary hardware and software components to implement the methods and techniques described 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 necessary computer components (e.g., processors, storage devices, input / output devices, displays, etc.) to control the actuators and implement the various system control processes discussed herein. The integrated sample staining and processing system 100 may further include sensors or data collection devices required to implement the various systems and control processes discussed herein, such as cameras, optical devices, temperature measurement devices, pressure sensors, force sensors, etc.

[0032] Figure 2A A sample staining method consistent with its embodiments is shown. Figures 2B-2C Schematic diagrams of some aspects of staining method 1000 are provided. Staining method 1000 can be performed according to the systems, techniques, and apparatus disclosed herein. The systems, techniques, and apparatus discussed herein provide examples illustrating the operation and performance of elements of staining method 1000 and are not exclusive. Therefore, while staining method 1000 can be implemented by employing staining cartridge 303 (or any other staining cartridge discussed herein) in conjunction with integrated sample processing system 100, this disclosure is not limited to this combination. The steps and operations of staining method 1000 described herein can be implemented in any suitable order and any suitable combination. In various embodiments, some or all steps and operations of staining method 1000 can be implemented using staining cartridge 303 and manual processing techniques. In other examples, integrated sample processing system 100 can perform any choice of steps and operations of staining method 1000, alone or in any combination, with or without using staining cartridges. The steps and operations of staining method 1000 can be performed independently, or in any suitable combination with other steps and operations of staining method 1000, and in any suitable combination with manual and automated processing techniques.

[0033] The dyeing method 1000 operates by distributing a dyeing solution onto a substrate using a dyeing surface. These are shown in... Figure 2B and Figure 2CIn this context, substrate 201 is the substrate on which the operator intends to stain, and typically includes a sample (e.g., a biological sample) disposed thereon for staining. Substrate 201 can be, for example, a sample slide made of a suitable material (e.g., glass, etc.). Substrate 201 is not limited to a sample slide and can include any structure suitable for staining the sample, including cell culture dishes, microplates, etc. Staining surface 204 is positioned opposite one side of substrate 201 on which the sample is disposed. Substrate 201 and staining surface 204 can be flat or substantially flat (e.g., with an average deviation from the average profile of less than 50 micrometers) and / or can have surface features (e.g., concave surfaces, convex surfaces, ridges, wells, bumps, etc.) to facilitate staining agent distribution and / or sample placement. Staining surface 204 can be positioned on any suitable structure, such as a flat surface (e.g., a coverslip), a disposable box, a durable manifold, etc. During dyeing, the substrate 201 and the dyed surface 204 are separated by a gap 290, which may be referred to herein as a vertical gap and / or capillary gap. The term "vertical" as used with respect to these two structures does not require a specific orientation and is chosen to indicate the relative spacing between them. For example, as used herein, when the substrate 201 and the dyed surface 204 are arranged side-by-side, the gap 290 will still be considered and referred to as a "vertical gap." During dyeing, the gap 290 can be "maintained" by suitable mechanical or electromechanical means as discussed herein. In various embodiments, the spacing of the gap 290 can vary in one or more dimensions across the overlap 291. For example, as described below... Figures 4A-4JAs discussed, the gap 290 may have a relatively small value at the center of the overlap 291 and a relatively large value at the edges of the overlap 291. This may be referred to as the gap profile. "Maintaining" the gap may include maintaining the gap at a specific predetermined distance, within tolerances of, for example, 1%, 5%, or 10%, or having a specific gap profile with distances varying in 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 disrupting the surface tension of the liquid disposed therein. Thus, a maintaining gap can be achieved while allowing changes in the size of the gap—without causing loss of capillary action or surface tension. The stained surface 204 and the substrate 201 may be arranged close to each other to establish the overlap 291, also referred to as a lateral or horizontal overlap. The terms "lateral" and "horizontal" to refer to the overlap 291 are intended only for relative examples. The nature of the overlap 291 does not change with different orientations. The overlapping portion 291 represents a common area where the substrate 201 and the dyeing surface 204 overlap or occupy the same region (albeit on different planes) when viewed along an axis perpendicular to the substrate 201 and the dyeing surface 204. The overlapping portion 291 can also be understood as a region within which orthogonal vectors originating from the substrate 201 and extending toward the dyeing surface 204 will intersect the dyeing surface 204. The overlapping portion 291 (which is a region) and the gap 290 (which is a distance) together establish the dyeing space 299, which represents the volume between the overlapping areas of the substrate 201 and the dyeing surface 204.

[0034] During staining, chromosome volume 299 is filled with staining solution. In the embodiments disclosed herein, the staining solution within chromosome volume 299 is constrained only by the substrate 201, the staining surface 204, and the combination of the adhesive forces between the staining solution and the substrate 201 and the staining surface 204, and the surface tension of the staining solution. Therefore, chromosome volume 299 may have one or more meniscus edges 293. The meniscus edges 293 are defined by the periphery of the region of the overlap 291 and are not constrained by walls or other physical structures. It is not required that the entire periphery of chromosome volume 299 be defined by meniscus edges 293. In various embodiments, at least a portion of chromosome volume 299 may be defined by walls or other physical structures, while the remaining portion is defined by one or more meniscus edges 293. Thus, chromosome volume 299 may be larger (e.g., 1%, 5%, 10%, etc.) than the rectangular prism defined by the overlap 291 and the gap 290 by the volume of the liquid meniscus or bulge appearing at the meniscus edges 293 of chromosome volume 299.

[0035] During staining distribution, the overlap 291 increases, as... Figure 2CAs shown, this increases the chromosome volume 299. As the overlap 291 increases, additional staining solution is supplied to the chromosome volume 299, proportional to the increase in the volume of the chromosome volume 299. In various embodiments, the additional staining solution is received at the chromosome volume 299 via one or more meniscus edges 293. The additional staining solution may already be present (e.g., pre-deposited) on the staining surface 204 and / or may be deposited continuously at the meniscus edges 293 on the staining surface 204.

[0036] The following text is about Figure 2A and Figure 2B (It provides general examples of some of these structures and features) and other figures with specific examples of the structures and features provided to describe the operation of method 1000 in more detail.

[0037] In operation 1002, the dyeing method 1000 includes disposing of a dyeing solution or a portion of a dyeing solution on a dyeing surface. Dyeing solution deposition may include disposing of at least one dyeing solution on the dyeing surface. As described herein, the dyeing surface may be dyeing surface 204, or the dyeing surface 304 of a dyeing cassette (e.g., dyeing cassette 303). Figure 3A (as shown in the diagram) or any other suitable staining surface. The staining surface may also include other structures not part of the staining box, such as coverslips or any other surface. Arranging the staining solution on the staining surface includes steps and techniques suitable for applying a fluid (e.g., a staining fluid) to the staining surface. Such steps and techniques may include, but are not limited to, arranging, dripping, sprinkling, spraying, or otherwise depositing the arranged staining solution onto the staining surface in preparation for staining. Furthermore, the arrangement of the staining solution may include the use of a staining agent delivery device, and / or a port, and / or any other suitable means. As described herein, the staining agent delivery device may include staining agent delivery device 302 (e.g., Figure 3A As shown), the port may include port 406 (e.g., Figure 4A (As shown in the diagram). As discussed herein, staining solution placement can be performed manually by an operator or by an integrated sample handling system. As described herein, staining surfaces and staining cartridges represent exemplary ways to facilitate staining solution placement, but are not essential. Staining solution placement operations can be performed by any manually or automatically operated structure or device capable of placing the liquid. For example, staining agent delivery device 302 (e.g., Figure 3A As shown, the dye solution can be disposed of by physical force (e.g., applying sufficient pressure within the dye delivery device 302 to dispose of a predetermined amount of dye solution on the dyeing surface) or by natural force (e.g., allowing the dye delivery device 302 to gravity-feed the dye solution onto the dyeing surface). In a further embodiment, the dye can be delivered via capillary action. Other methods and techniques for disposing of the dye solution are described below.

[0038] In operation 1004, the dyeing method includes arranging a substrate near the dyeing surface. The substrate (e.g., substrate 201) may be arranged to establish an overlap (e.g., overlap 291) with the dyeing surface (e.g., dyeing surface 204). As described herein, the substrate may be a dyeing plate (e.g., Figure 3A As shown herein, it may include, for example, a sample slide. As discussed herein, staining plate arrangement can be performed manually by an operator or by an integrated sample handling system (e.g., by its actuators). The staining plates, staining surfaces, and staining cassettes described herein represent exemplary ways to facilitate staining plate arrangement, but are not essential. Staining plate arrangement operations can be performed by any manual or automated method capable of positioning the staining plate and staining surface 304 in a location with vertical gaps and horizontal overlap (e.g., Figure 3A , Figure 4A , Figure 5A and Figure 6A The dyeing plate arrangement operation can be performed by any structure or device (as shown in the diagram). The dyeing plate arrangement operation can further be performed by any manually or automatically operated structure or device capable of controlling or maintaining the horizontal overlap between the dyeing plate and the dyeing surface, and may further include controlling the rate of change of the horizontal overlap between the dyeing plate and the dyeing surface. Other methods and techniques for dyeing plate arrangement are described below.

[0039] In operation 1006, staining method 1000 includes maintaining a gap between a substrate and a staining surface. The gap (e.g., gap 290) and overlap (e.g., overlap 291) establish or define chromosome volumes (e.g., chromosome volumes 299). The established chromosome volumes may have one or more meniscus edges (e.g., meniscus edge 293). As discussed herein, maintaining the gap can be performed manually by an operator or by an integrated sample processing system. As described herein, structures for maintaining the gap between the staining plate and the staining surface may include gap-maintaining structures. As used herein, "gap-maintaining structure" refers to a structural element configured to maintain a specific gap between the staining plate (e.g., substrate) and the staining surface. In one embodiment, one or more pairs of ledges 308 (e.g., Figure 3A (As shown in the diagram) A gap-maintaining structure can be provided. Lug 308 is configured to support the dyeing plate from below and hold the dyeing plate fixed or at a predetermined distance above the dyeing surface. In a further embodiment, the gap-maintaining structure can be configured to support the dyeing plate from below and hold the dyeing plate fixed or at a predetermined distance below the dyeing surface. Such a gap-maintaining structure is shown in... Figure 9 middle, Figure 9A dyeing structure 900 is shown, comprising a dyeing surface 904 and a gap-maintaining structure defined by one or more lugs 908. The lugs 908 are configured to support a dyeing plate (e.g., a substrate) 901 and position the dyeing plate 901 at a predetermined or fixed distance below the dyeing surface 904 to establish a gap 290. It will be understood that the features of the dyeing structure 900 can be combined with any other embodiments disclosed herein. Although regarding... Figure 9 and Figure 3A The term "lug" is used, but the gap-maintaining structure is not limited to this and can include any physical structure capable of supporting the dyeing plate. In a further embodiment, the gap can be maintained by a gap-maintaining device configured to hold the dyeing plate at a predetermined or fixed distance from the dyeing surface. Gap-maintaining devices include, but are not limited to, robot actuators and other mechanical devices such as lead screws, levers, etc. In a further embodiment, the gap can be maintained by a combination of gap-maintaining structures and devices; for example, the gap-maintaining structure may include a gantry movable via the action of a robot actuator. Maintaining the gap between the dyeing plate and the dyeing surface includes steps, techniques, structures, and devices adapted to maintain the gap between the dyeing plate and the dyeing surface to avoid contact between the dyeing plate and the dyeing surface and to maintain a sufficient gap (e.g., neither too large nor too small) to maintain capillary action (e.g., creating and maintaining a capillary gap) between the dyeing plate and the dyeing surface. Such steps and techniques may include, but are not limited to, controlling or maintaining the horizontal overlap between the dyeing plate and the dyeing surface, controlling the rate of change of the horizontal overlap between the dyeing plate and the dyeing surface, controlling or maintaining the vertical gap between the dyeing plate and the dyeing surface, or otherwise controlling the dyeing plate and the dyeing surface and controlling or maintaining the horizontal overlap and vertical gap between them. The dyeing plate, dyeing surface, lug, and dyeing box described herein represent exemplary ways of facilitating gap maintenance operations, but are not essential. Gap maintenance operations can be performed by any manually or automatically operated structure or device capable of maintaining the relative positioning between the dyeing plate and the dyeing surface and / or controlling their relative movement. For example, in addition to the structures described herein configured to maintain the gap between the dyeing surface and the dyeing plate, one or more robotic actuators may also be configured to maintain the gap between the dyeing surface and the dyeing plate. Other methods and techniques for maintaining the dyeing plate close to the dyeing surface are described below.

[0040] In operation 1008, the staining method includes distributing a staining solution onto a substrate. In various embodiments, distributing the staining solution onto the substrate can be performed by increasing the overlap between the staining surface and the substrate (thereby increasing the chromosome volume), and as the overlap is increased, filling the chromosome volume with the staining solution. The distribution of the staining solution is shown in... Figure 2CAs described herein, distributing a dye solution includes the steps and techniques described herein suitable for distributing a fluid or liquid (e.g., a dye solution) onto a substrate. Such steps and techniques may include, but are not limited to, sliding, sliding, spreading, spraying, smearing, or otherwise distributing the disposed dye solution onto the substrate. Furthermore, dye solution distribution may also include using a dye delivery device 302 (e.g., [missing information - likely a device name]). Figure 3A As shown), port 406 (for example, Figure 4A (as shown in the diagram) and any other suitable distribution device. The dyeing solution distribution operation 1008 may also include controlling the flow of the dyeing solution arranged on the substrate.

[0041] According to the various embodiments herein, troughs (e.g., in a dyeing box) can be used. Figure 3A The grooves 307 around the dyeing surface 304 of the dyeing box 303 shown are used to control the flow of dyeing solution arranged on the substrate. The grooves are positioned such that the dyeing solution will remain on the dyeing surface (e.g., Figure 3A The staining solution is applied to the staining surface 304 shown herein, and the valleys are avoided due to adhesion to the substrate (staining plate), adhesion to the staining surface, and the surface tension of the staining solution. Furthermore, according to the various embodiments herein, controlling the flow of the staining solution arranged on the substrate can also be achieved by using hydrophobic and / or hydrophilic coatings on various surfaces, including but not limited to the substrate (staining plate), staining surfaces, and / or valleys in the staining cassette. As discussed herein, staining solution distribution can be performed manually by an operator or by an integrated sample handling system. The staining plates, staining surfaces, and staining cassettes described herein represent exemplary methods for facilitating staining solution distribution, but are not essential. Staining solution distribution operations can be performed by any manually or automatically operated structure or device capable of distributing liquids. Other methods and techniques for distributing staining solutions are described below.

[0042] Figures 3A-3K The various stages of the process of distributing the staining solution onto a staining plate are shown. According to the illustrated embodiment, Figures 3A-3K A staining plate 301, a staining agent delivery device 302, and a staining cassette 303 are shown. As described herein, the staining plate 301 receives a sample. Arranging the sample on the staining plate 301 includes steps and techniques suitable for spreading, smearing, or otherwise distributing the deposited sample on the staining plate 301 to prepare for staining. Furthermore, as described herein, the staining agent delivery device 302 may also include a tubular structure for delivering the staining solution. The staining agent delivery device 302 is not limited to... Figures 3A-3K The shape shown, and any suitable structure for (e.g., as described above with respect to operation 1002 of method 1000) arranging the dye solution on the dyeing surface, are within the scope of the claimed invention.

[0043] The dyeing cassette 303 may include any or all of the following: a base 391 and a pair of lugs 308 forming a lug structure, a dyeing surface 304 (e.g., a surface that receives the disposed dyeing solution), a port 306, a trough 307, and a discharge portion 305. The base 391 is configured such that other features of the dyeing cassette 303 are disposed thereon or within it. As described herein, the lugs 308 are sized (e.g., in height and width) sufficient to create a gap 312 between the dyeing surface 304 and the dyeing plate 301, and maintain the gap 312 (e.g., as described above with respect to operation 1004 of method 1000) to create a horizontal overlap 311 as the dyeing plate 301 slides past the lugs 308. Direct contact between the dyeing plate 301 and the lugs 308 is not required, as the dyeing plate 301 may be in a holder or other device. Therefore, the top of the lugs 308 is positioned at a predetermined distance above at least a portion of the dyeing surface 304. As discussed below, the staining surface 304 may include one or more features to facilitate the distribution of the staining solution, and may therefore be characterized by a gap profile. A predetermined distance sets the height of the vertical gap 312, which may be a capillary gap. The vertical gap 312 (and therefore the height of the top of the lug 308 above the staining surface 304) may be maintained at a height of less than 350 micrometers, less than 300 micrometers, less than 250 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers. The lug 308 is not limited to... Figures 3A-3K The shape shown, 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 have substantially the same height over the overlap 311 (e.g., within 5%), and the staining surface 304 and the staining plate 301 may be substantially parallel. In a further embodiment, as described below, the staining surface 304 may include raised features, such as raised structures 310 with varying heights of the vertical gap 312. The overlap 311 and the gap 312 together define a chromosome volume 399.

[0044] As described herein, the groove 307 can be arranged within the dyeing box 303. According to various embodiments, the groove 307 is sized (e.g., in height and width) sufficiently and is positioned such that the dyeing solution will remain on the dyeing surface 304 and is avoided by adhesion to the substrate (dyeing plate), adhesion to the dyeing surface, and the surface tension of the dyeing solution. Figure 3A As shown, the groove 307 can be configured to surround or partially surround the dyeing surface 304 with a surface lower than the height of the dyeing surface 304. The groove 307 is not limited to... Figures 3A-3KThe shape shown, and any suitable structure for controlling the distribution of the dye solution on the dyeing 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 various embodiments, the cassette 303 does not require the groove 307. In various embodiments, the substrate 301 may not extend to the edge of the staining surface 304. Therefore, as the staining surface 304 continues to extend, the meniscus edge of the chromosome product 399 may be established at the edge of the substrate 301. In some embodiments, the staining surface 304 may extend to the lug 308, and the meniscus edge of the chromosome product 399 (e.g., both edges) may be established only at the end of the substrate 301, while the lateral edge of the chromosome product 399 is defined by the lug 308. In further embodiments, as discussed throughout, the cassette 303 may not include the lug 308, and the gap 312 may be maintained by an actuator (e.g., a robotic actuator).

[0046] According to the embodiments described herein, the discharge unit 305 can actively (e.g., by generating a vacuum via a pump, sufficient to actively pull the dyeing solution away from the dyeing surface 304 through the discharge unit 305) or passively (e.g., by the discharge unit 305 passively drawing the dyeing solution away from the dyeing surface 304) via a conduit 309 connected to port 306 to draw fluid (e.g., dyeing solution). According to other embodiments herein, an absorbent pad (not shown) can remove the dyeing solution from the dyeing surface 304. The absorbent pad can be positioned within a cartridge 303, for example, in a compartment connected to the conduit 309. The suction or vacuum drawn from the discharge unit 305 can be used to draw excess dyeing solution into the compartment in the conduit 309, where it can be absorbed by the absorbent pad. In this way, all excess dyeing solution can remain in the cartridge 303, thereby keeping the system clean.

[0047] The dyeing cartridge 303 may further include a protrusion 310 disposed on the dyeing surface 304. As shown, the protrusion 310 creates a tapered, tapering, or volcano-like surface around a port 306 (e.g., an inlet or outlet port) of the dyeing surface 304, sufficient to draw fluid (e.g., dyeing solution) toward the port 306 due to increased capillary action as the capillary gap decreases. In various embodiments, the protrusion 310 may be configured to extend a sufficient distance away from the port 306 to prevent air from entering the port 306 as the dyeing solution exits through the port. A sufficient distance may include greater than 2 mm, greater than 3 mm, greater than 4 mm, and / or greater than 5 mm. The gap profile of the gap 312 may be defined by a constant height portion and a protrusion portion, wherein the constant height portion provides a constant height for the gap profile, while the protrusion portion provides a decreasing height for the gap profile at the protrusion 310. The gap 312 may narrow from most of the dyeing surface 304 toward the apex of the protrusion 310 where the port 306 is located. When port 306 is an inlet port, the protrusion 310 can assist in pushing air bubbles away from the dyeing surface region 304. Furthermore, when port 306 is an outlet port, the protrusion 310 helps retain liquid around and flowing into port 306, and prevents port 306 from being exposed to air (e.g., when port 306 is exposed to air, air prevents liquid from being drawn into port 306, thereby preventing further discharge). Moreover, the design of port 306 is not limited to that shown in the figures, and the embodiments described herein include shapes more complex than a simple circular shape. For example, a slit shape can guide the flowing dye solution across a greater distance. In various embodiments, port 306 may include a linearly arranged series of orifices connected to conduit 309 via a manifold. A slit-shaped port 306 or one including a series of orifices may extend across the dyeing 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 dyeing surface 304.

[0048] like Figures 3A-3EAs shown, according to operation 1002 of method 1000, the dyeing solution is placed on the exposed dyeing surface 304 via the dye delivery device 302. Placing it on the exposed dyeing surface 304 provides an opportunity for any air bubbles that may be present in the dye delivery device 302 to escape into the environment. The dyeing solution can be arranged such that it is directed to an unconstrained environment into which air bubbles can freely escape. An unconstrained environment can include any environment adjacent to the dyeing solution that does not prevent air bubbles from escaping from the liquid. Examples of an unconstrained environment can include an atmospheric or localized gas environment with pressure and composition that does not restrict the movement of air bubbles from the liquid. Once air bubbles or air gaps enter the gap 312, they can be difficult to remove, resulting in portions of the substrate 301 either not being in contact with the dye or having reduced dye contact.

[0049] According to the illustrated embodiment, according to operation 1004 of method 1000, the dyeing plate 301, the dye delivery device 302, and the dyeing cassette 303 are moved relative to each other, such that the dyeing plate 301 is arranged near the dyeing surface 304 of the dyeing cassette 303, and according to operation 1006 of method 1000, the lug 308 maintains a gap 312 (e.g., capillary gap) between the dyeing plate 301 and the dyeing cassette 303. Furthermore, according to various embodiments, when the dyeing cassette 303 moves, the dye delivery device 302 may remain stationary (e.g., the dyeing cassette 303 moves below the stationary dye delivery device 302). According to other embodiments, the dyeing cassette 303 is stationary, while the dye delivery device 302 is movable (e.g., the dye delivery device 302 moves above the stationary dyeing cassette 303). In still other embodiments, both the dye delivery device 302 and the dyeing cassette 303 may be movable. Figures 3F-3I It shows Figures 3A-3E The same movement is shown, but in cross-sectional view and from a different angle, so that the protrusion structure 310 and the conduit 309 are better shown with respect to the dyeing surface 304, the port 306 and the dyeing plate 301.

[0050] like Figure 3J and Figure 3K As shown, according to operation 1008 of method 1000, the staining agent delivery device 302 is distributing the staining solution onto the staining surface 304. As shown, the staining plate 301 (e.g., with a sample, not shown) is swept or moved across the staining surface 304. According to the embodiments described herein, the staining solution fills the gap 312 (e.g., capillary space) between the staining plate 301 and the staining surface 304 by entering the chromosome volume 399. In various embodiments, the staining solution enters the chromosome volume 399 at its meniscus edge (not shown).

[0051] like Figures 3A-3EAs shown, before the dyeing plate 301 is swept, the dye delivery device 302 can deposit a predetermined amount of dye solution on the dyeing surface 304. When the dyeing plate 301 completely covers the dyeing surface 304, the predetermined amount of dye solution can be selected according to the volume of the gap 312. The predetermined amount of dye solution can be equal to this volume. In various embodiments, the dye delivery device 302 can deposit a predetermined amount of dye solution during the sweeping of the dyeing plate 301, and can deposit the dye solution onto the dyeing surface 304 in front of the moving dyeing plate 301. In a further embodiment, the dye delivery device 302 can deposit a predetermined amount of dye solution onto the dyeing plate 301 at the junction of the dyeing surface 304 and the dyeing plate 301, for example, directly into the gap 312 at its meniscus edge.

[0052] The embodiments described herein may further include various staining agents. For example, according to Figures 3A-3K The embodiments shown, including those involving various dyes, include removing the dye solution from the dyeing surface (e.g., via port 306, through conduit 309, and exiting discharge 305) and restoring the dye cartridge 303 and dye delivery device 302 to their original positions. Figure 3A The position shown is indicated. Once the position between the staining cartridge 303 and the staining agent delivery device 302 is reset, a second liquid (e.g., staining solution) is added (e.g., as described above and shown in the operation according to method 1000). In this way, any amount of staining solution, fixative, and / or cleaning solution can be introduced. In a further embodiment, port 306 can be used as an input / output port. The first staining solution can be removed via port 306, and the second staining solution can be introduced into gap 312 via port 306. Potentially, the second staining solution can be removed via port 306 to introduce additional liquids (staining, cleaning, fixing, etc.). In this way, any amount of liquid can be introduced and removed.

[0053] Figures 4A-4J The various stages of the process of distributing the staining solution onto a staining plate are illustrated. According to one embodiment, Figures 4A-4J A staining plate 301 and a staining cassette 403 are shown. As described herein, the staining plate 301 can receive a sample. Arranging a sample on the staining plate 301 may include steps and techniques suitable for preparing the sample for staining by scattering, smearing, or otherwise distributing the deposited sample on the staining plate 301.

[0054] According to various embodiments, the dyeing cassette 403 may include any or all of the following: a pair of lugs 408 forming a lug structure, a dyeing surface 404 (e.g., a surface that receives the disposed dyeing solution), a port 406, a trough 407, and a discharge portion 405. As described herein, the lugs 408 are sized (e.g., in height and width) to create and maintain a gap 412 between the dyeing surface 404 and the dyeing plate 301 as the dyeing plate 301 slides past the lugs 408 (e.g., as described above with respect to operation 1004 of method 1000). In various embodiments, the dyeing surface 404 may be parallel to the dyeing plate 301 and thus have a constant gap 412 spanning its entire surface. In such embodiments, the top of the lugs 408 may be positioned at a predetermined distance above the dyeing surface 404. The predetermined distance defines the height of the vertical gap 412. The vertical gap 412 (and therefore the height of the top of the lug 408 above the dyeing surface 404) can be maintained at a height of less than 350 micrometers, less than 300 micrometers, less than 250 micrometers, less than 200 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers, and can be a capillary gap. In a further embodiment, as discussed below, the ramp 410 arranged on the dyeing surface 404 can create a variable gap 412 or gap profile. The gap profile of the gap 412 can be defined by the ramp 410, which provides a variable gap. The lug 408 is not limited to... Figures 4A-4J The shape shown, and any suitable structure used (e.g., as described above with respect to operation 1006 of method 1000) to maintain the gap 412 between the dyeing surface 404 and the dyeing plate 301, is within the scope of the claimed invention. Furthermore, as described herein, a groove 407 is arranged within the dyeing cassette 403. According to various embodiments, the groove 407 is sized (e.g., in height and width) sufficiently and is positioned such that the dyeing solution 420 will remain on the dyeing surface 404 and avoid the groove 407 due to the surface tension of the dyeing solution 420 and its adhesion to the contacted surface. The groove 407 may be configured to surround or partially surround the dyeing surface 404 with a lower surface. The groove 407 is not limited to... Figures 4A-4J The shape shown, and any suitable structure for controlling the distribution of the dye solution on the dyeing 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 various embodiments, the cassette 403 does not require the groove 407. In various embodiments, the substrate 401 may not extend to the edge of the staining surface 404. Therefore, as the staining surface 404 continues to extend, the meniscus edge of the chromosome product 499 may be established at the edge of the substrate 401. In some embodiments, the staining surface 404 may extend to the lug 408, and the meniscus edge of the chromosome product 499 (e.g., both edges) may be established only at the end of the substrate 401, while the lateral edge of the chromosome product 499 is defined by the lug 408. In further embodiments, as discussed throughout, the cassette 403 may not include the lug 408, and the gap 412 may be maintained by an actuator (e.g., a robotic actuator).

[0056] According to the embodiments described herein, the discharge unit 405 can actively (e.g., by generating a vacuum via a pump, sufficient to actively pull the dyeing solution 420 away from the dyeing surface 404 through the discharge unit 405) or passively (e.g., the discharge unit 405 passively draws the dyeing solution 420 away from the dyeing surface 404) via the conduit 409 to draw fluid (e.g., dyeing solution 420). According to other embodiments herein, an absorbent pad (not shown) can remove the dyeing solution 420 from the dyeing surface 404. The absorbent pad can be positioned within the cartridge 403, for example, in a compartment connected to the conduit 409. The suction or vacuum drawn from the discharge unit 405 can be used to draw excess dyeing solution into the compartment in the conduit 409, where it can be absorbed by the absorbent pad. In this way, all excess dyeing solution can remain within the cartridge 403, thereby keeping the system clean.

[0057] The dyeing surface 404 may further include a ramp 410. The ramp 410 may provide a tapered or tapered surface around a port 406 (e.g., an inlet or outlet port) of the dyeing surface 404, sufficient to draw fluid (e.g., dye solution 420) toward the port 406 via capillary action. The ramp 410 may cover the entire dyeing surface 404 and / or may cover only a portion of the dyeing surface 404. The ramp 410 may be used to create a variable gap 412. When the port 406 is an inlet port, the ramp 410 assists in pushing air bubbles out of the dyeing surface region 404. Furthermore, when the port 406 is an outlet port, the ramp 410 assists in retaining liquid around and flowing into the port 406, and preventing the port 406 from being exposed to air (e.g., when the port 406 is exposed to air, air prevents liquid from being drawn into the port 406). In a further embodiment, the port 406 may simultaneously function as both an inlet and an outlet. Furthermore, the design of port 406 is not limited to that shown in the figures, and the embodiments described herein include shapes more complex than a simple circular shape. For example, a slit-shaped port guides the flow of staining solution 420 across a greater distance. In various embodiments, port 406 may include a linearly arranged series of orifices connected to conduit 409 via a manifold. Slit-shaped port 406 or port 406 including a series of orifices may extend across 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 staining surface 404.

[0058] In various embodiments, for example, such as Figures 4A-4J As shown, port 406 may be located approximately at the center of the stained surface 404. Figures 4G-4J As shown, the ramp 410 can form a tapered or volcano-like structure on the dyeing surface 404. In various embodiments, the protrusion 410 can be configured to extend a sufficient distance away from the port 406 to prevent air from entering the port 406 when the dyeing solution exits through the port. A sufficient distance can include a distance greater than 2 mm, greater than 3 mm, greater than 4 mm, and / or greater than 5 mm. In one embodiment, the cone can be a uniform cone extending from the port 406 to the edge of the dyeing surface 404. Therefore, the slope of the cone is gentler in the long direction of the dyeing surface and steeper in the short direction of the dyeing surface 404.

[0059] exist Figure 4A and Figure 4B In this process, according to operation 1004 of method 1000, the staining plate 301 can be arranged near the staining surface 404 of the staining cassette 403 to establish an overlap 411. The overlap 411 and the gap 412 together establish or define a chromosome volume 499. In various embodiments, the overlap 411 can span the entire staining surface 404 before any staining solution is deposited. Figures 4B-4FAs shown, according to operation 1002 of method 1000, the dyeing solution 420 is disposed on the dyeing surface 404 through port 406. According to the various embodiments herein, port 406 can be operated as both an inlet and an outlet, such that the dyeing solution 420 (and subsequent liquids in the dyeing process) is pumped in and out of port 406. Figures 4G-4J It shows Figures 4B-4F The same movement of the staining solution 420 is shown, but in cross-sectional view and from a different angle, thus better showing the bevel 410 and conduit 409 with respect to the staining surface 404, port 406, and staining plate 401. As described herein, according to operation 1006 of method 1000, the lug 408 maintains the gap 412 between the staining plate 401 and the staining surface 404. Furthermore, according to various embodiments, the staining surface 404 may not be parallel to the lug 408 (and therefore not parallel to the staining plate 301). Instead, the staining surface 404 may be tapered due to the bevel 410, such that the edge of the staining surface is farther from the staining plate 301 than the center point (e.g., near port 406). Thus, the gap 412 is maintained, but gradually decreases as the staining surface 404 tapers. The gap 412 is maintained and does not disappear. However, the size of the gap 412 varies depending on the taper of the bevel 410 of the staining surface 404. In various embodiments, at the edge of the inclined surface 410, the gap 412 may be less than 350 micrometers, less than 300 micrometers, less than 250 micrometers, less than 200 micrometers, less than 150 micrometers, less than 100 micrometers, or less than 50 micrometers, and may or may not coincide with the edge of the dyeing surface 404. In various embodiments, the gap 412 at the location of the port 406 may be less than 300 micrometers, less than 250 micrometers, less than 200 micrometers, less than 150 micrometers, less than 100 micrometers, less than 50 micrometers, or less than 25 micrometers. The structure of the dyeing cassette 403 may distribute the dyeing solution 420 (e.g., according to operation 1008 of method 1000) such that the dyeing solution 420 is self-centered on the port 406 and prevents air from being drawn into the port 406 when the dyeing solution 420 is pumped out.

[0060] The embodiments described herein may further include various staining solutions. For example, according to Figures 4A-4J The embodiments shown, including those involving multiple dyeing solutions, may include removing dyeing solution 420 from the dyeing surface 404 (e.g., via port 406, through conduit 409 and out of discharge 405) and adding a second dyeing solution (e.g., another liquid dye, cleaning agent, fixative, etc.) to the gap 412 between the dyeing surface 404 and the dyeing plate 401 (e.g., as described above and shown in the operation according to method 1000). As described herein, the addition and extraction of fluids can occur very rapidly. For example, according to Figures 4A-4JIn the embodiments shown, adding and removing fluid in any direction may take 1 second or less, 2 seconds or less, 5 seconds or less, or 10 seconds or less. The ability to rapidly introduce and remove fluid from chromosome volume 499 provides the advantage of more uniform staining.

[0061] Figure 5A Figure 5V illustrates the various stages of the process of distributing the dye solution onto the substrate. According to the illustrated embodiment, Figure 5A Figure 5V illustrates a staining plate 301, a staining agent delivery device 502, and a staining cassette 503. As described herein, the staining plate 301 receives a sample. Arranging the sample on the staining plate 301 may include steps and techniques adapted to spread, smear, or otherwise distribute the deposited sample across the staining plate 301 in preparation for staining. As described herein, the staining agent delivery device 502 may include one or more tubular structures (e.g., 502A-502D) for delivering the staining solution. The staining agent delivery device 502 is not limited to... Figure 5A - The shape shown in Figure 5V, and any suitable structure for (e.g., as described above with respect to operation 1002 of method 1000) arranging the dye solution on the dyeing surface, are within the scope of the claimed invention.

[0062] The dyeing cassette 503 may also include any or all of the following: a pair of lugs 508 forming a lug structure, a dyeing surface 504 (e.g., a surface for receiving the arranged dyeing solution), a port 506, a trough 507, and a discharge portion 505. The lugs 508 may be structurally and functionally similar to lugs 308 and 408. The dyeing surface 504 may be structurally and functionally similar to dyeing surfaces 304 and 504. The port 506, trough 507, and discharge portion 505 may be structurally and functionally similar to ports 306 / 406, troughs 307 / 407, and discharge portions 305 / 405, respectively, including the addition of an absorbent pad. Therefore, the cassette 503 may incorporate the dyeing surface 504, which has a port 506 at one end surrounded by a protruding structure 510, similar to the protruding structure of the cassette 303, and may have a tapered, tapered, or volcano-shaped surface. In another embodiment (not shown), the case 503 may incorporate a dyeing surface 504, which has a port 506 at one end surrounded by a protruding structure 510, similar to the protruding structure of the case 403. In some embodiments, the case 503 may not include the features described above. Figures 3A-4J The described trough 507.

[0063] like Figures 5A-5CAs shown, the first staining solution can be deposited onto the staining surface 504 via the staining agent delivery device 502. The staining plate 501 can be advanced relative to the staining surface 504 to establish an overlap 511 and distribute the first staining solution within the gap 512. The overlap 511 and the gap 512 together establish or define a chromosome volume 599. As the staining plate 501 is advanced on the staining surface 504, the overlap 511 (and the chromosome volume 599) increases. According to one embodiment, the staining plate 301, the staining agent delivery device 502, and the staining cassette 503 can be moved relative to each other such that, according to operation 1004 of method 1000, the staining plate 301 is positioned near the staining surface 504 of the staining cassette 503, and according to operation 1006 of method 1000, the lug 508 maintains the gap 512 between the staining plate 301 and the staining cassette 503. 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 protrusion structure 510. As the staining plate 501 is advanced, the staining agent delivery device 502 may deliver staining solution to the meniscus edge of the chromosome volume 599 at a rate corresponding to the increasing size of the chromosome volume 599. Furthermore, according to various embodiments, the staining agent delivery device 502 may remain stationary while the staining cassette 503 is movable (e.g., the staining cassette 503 moves below the stationary staining agent delivery device 502). According to other embodiments, the staining cassette 503 may be stationary while the staining agent delivery device 502 may be movable (e.g., the staining agent delivery device 502 moves above the stationary staining cassette 503).

[0064] After the first staining solution is distributed, the staining cartridge 503 can be operated in various ways. For example... Figures 5D-5F As shown, additional staining solutions (e.g., liquid staining agents, fixatives, cleaning agents, etc.) can be distributed to the open end (e.g., the meniscus edge) of the gap 512 at one end of the staining plate 301. Because the meniscus edge leads to an unconstrained environment, any accumulated air bubbles can be released into the unconstrained environment. Additional staining solutions can be distributed when port 506 is operated to remove previous staining solutions. If the rate of removal of old staining solutions is the same as the rate of addition of new staining solutions, the new staining solutions will flow smoothly through the gap 512 without generating air bubbles due to interruption of the liquid flow. In this way, any number of staining solutions can be added and removed sequentially. This distribution represents an example of distributing each additional staining solution according to operations 1002, 1004, 1006, and 1008.

[0065] Figure 5G-Figure 5J Alternative techniques for delivering additional staining solutions are shown. For example... Figure 5GAs shown, the first staining solution can be removed via port 506. To assist removal, the staining plate 301 can be pulled back or slid back as shown. Figure 5H The starting position is shown in Figure 51. Next, as shown in Figure 51 to... Figure 5J As shown, while the dyeing plate 301 is advanced on the dyeing surface 504, the second dyeing solution can be distributed by the dye delivery device 502. In this way, any amount of dyeing solution can be added and removed sequentially without the liquids touching each other. This distribution represents an example of distributing each additional dyeing solution according to operations 1002, 1004, 1006, and 1008.

[0066] Figures 5K-5N Shown from cross-sectional and side views Figures 5A-5C The steps are illustrated to further demonstrate the distribution of the staining solution on the staining plate.

[0067] Figures 6A-6H The various stages of the process of distributing the dye solution onto a substrate are shown. According to the illustrated embodiment, Figures 6A-6H A staining plate 301 and a staining cassette 603 are shown. As described herein, the staining plate 301 receives a sample. Arranging the sample on the staining plate 301 may include steps and techniques suitable for preparing the sample for staining by scattering, smearing, or otherwise distributing the deposited sample on the staining plate 301. The staining cassette 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 structures raised above a groove 607. Each staining surface 604A / B / C / D provides a surface isolated from the other staining surfaces 604A / B / C / D. The plurality of staining surfaces 604A / B / C / D may be square, rectangular, elliptical, or any other suitable shape, and is not limited thereto. Figure 6A - The number or shape shown in Figure 6N. For example, the multiple staining surfaces 604A / B / C / D can be larger or smaller, more or fewer in number, configured differently, and / or have different shapes. As described herein, the multiple staining surfaces 604A / B / C / D encompass any suitable structure for receiving the staining solution onto the staining surface (e.g., as described above with respect to operation 1002 of method 1000).

[0068] The dyeing box 503 may also include a groove 507 and a pair of lugs 508 forming a lug structure. The lugs 508 may be structurally and functionally similar to lugs 308 / 408 / 508. The grooves 507 may be structurally and functionally similar to grooves 307 / 407 / 507. Further...

[0069] like Figures 6A-6H As shown, according to operation 1002 of method 1000, the staining solution can be applied to each of the exposed staining surfaces 604A / B / C / D. Figures 6A-6E A perspective view of the dyeing box 603 is shown, while Figures 6F-6G Alternative cross-sectional views are shown. According to... Figures 6A-6H In the embodiments shown, various different staining solutions 620A / B / C / D can be deposited on multiple staining surfaces 604A / B / C / D. As shown, as an example of operations 1004 and 1008 according to method 1000, a staining plate 301 (e.g., with a sample, not shown) is swept across multiple staining surfaces 604A / B / C / D, and the staining solutions 620A / B / C / D deposited thereon stain, spread, or smear across the staining plate 301. According to the described embodiments, according to operation 1006 of method 1000, the gap 612 between the staining plate 301 and the multiple staining surfaces 604A / B / C / D is maintained and is less than 350 micrometers (or less than 300 micrometers, or less than 250 micrometers, or less than 200 micrometers, or less than 150 micrometers, or less than 100 micrometers, or less than 50 micrometers, or less than 25 micrometers).

[0070] Figures 6A-6H The application of various liquids (e.g., various staining solutions) to a staining plate 301 is illustrated. According to various embodiments, when an overlap 611 is established between the staining plate and one of the plurality of staining surfaces 604A / B / C / D, the staining plate 301 moves across a staining cassette 603 (e.g., on a lug 608) and contacts or otherwise interacts with the staining solution deposited on one of the plurality of staining surfaces 604A / B / C / D. The plurality of overlaps 611 and gaps 612 each establish a corresponding associated chromosome volume 699. For example, as... Figure 6C As shown, the dyeing plate 301 passes over the dyeing surface 604A and establishes an overlap 611 therewith, and when the dyeing agent or other liquid (e.g., dyeing solution 620 A) is pre-deposited on the dyeing surface 604A, the dyeing agent or other liquid is applied, spread, or otherwise distributed on the dyeing plate 301. Figure 6CAs further shown, the dyeing plate 301 then passes over the dyeing surface 604B, establishing an overlap 611 therewith. When a dye or other liquid (e.g., dyeing solution 620B) is pre-deposited on the dyeing surface 604B, the dye or other liquid is applied, spread, or otherwise distributed onto the dyeing plate 301 and replaces the dye or other liquid (e.g., dyeing solution 620A) applied to the dyeing plate 301 from the dyeing surface 604A. The dyeing plate 301 then passes over the dyeing surface 604C, establishing an overlap 611 therewith. When a dye or other liquid (e.g., dyeing solution 620C) is pre-deposited on the dyeing surface 604C, the dye or other liquid is applied, spread, or otherwise distributed onto the dyeing plate 301 and replaces the dye or other liquid (e.g., dyeing solutions 620A and 620B) applied to the dyeing plate 301 from the dyeing surfaces 604A and 604B. Figure 6D As shown, the dyeing plate 301 eventually passes over the dyeing surface 604D, establishing an overlap 611 therewith. While a dye or other liquid (e.g., dye solution 620D) is pre-deposited on the dyeing surface 604D, the dye or other liquid is applied, spread, or otherwise distributed onto the dyeing plate 301 and replaced from dyeing surfaces 604A, 604B, and 604C with the dyeing liquid applied to the dyeing plate 301. Therefore, different portions of the substrate 601 can be exposed to different dye solutions at any given time. Furthermore, in various embodiments, the dyeing surfaces 604A, 604B, and 604C can be of different sizes, which allows for adjustment of the amount of time any portion of the substrate 601 (and the sample thereon) is exposed to a given dye solution—if the substrate 601 is advanced at a constant rate. In other embodiments, the advancement rate of the substrate 601 can be variable and can include one or more pauses, accelerations, reversals, etc.

[0071] As mentioned above, Figure 6A The embodiment of Figure 6N does not require any mechanism to pump or otherwise move the liquid during the movement of the dyeing plate 301. Specifically, as shown, the liquid is pre-deposited on one or more of the dyeing surfaces 604A / B / C / D, and subsequently interacts with the dyeing plate 301 as it advances and interacts with the dyeing surfaces 604A / B / C / D.

[0072] Figures 7A-7D The various stages of the process of distributing the dye solution onto a substrate are shown. According to the illustrated embodiment, Figures 7A-7DThe illustration shows a dyeing cartridge 703 and a dye distribution device 702, one or more dye storage sections 709, one or more ports 706, a dyeing surface 704, and one or more inlets 716. The dye storage section 709 is a cavity within the dyeing cartridge 703 configured to store dyeing solution. The inlet 716 is an aperture providing passage to the dye storage section 709. The port 706 is an outlet aperture providing passage from the dye storage section 709 to the dyeing surface 704. In various embodiments, one or more of the ports 706 and inlets 716 may include removable covers such as adhesive foil, plunger tape, valve covers, flexible valve flaps, etc. Furthermore, as described herein, the dye distribution device 702 includes an air jet device 702A. The dye distribution device 702, air jet device 702A, one or more dye storage sections 709, one or more ports 706, and one or more inlets 716 together form an assembly for arranging and / or distributing liquid as described above with respect to operation 1002 of method 1000. The components and related parts of the dye distribution device 702 are not limited to Figures 7A-7D The shape shown, and any suitable structure used for (e.g., as described above with respect to operations 1002 and 1008 of method 1000) dispersing the dye solution onto the dyeing surface, are within the scope of the claimed invention. For example, the dye storage portion 709 is not limited to Figures 7A-7D The linear tubular shape shown, and any suitable structure for controlling the distribution of the dye solution on the dyeing surface 604 (e.g., a curved or meandering tubular structure within the dyeing cartridge 703), are within the scope of the claimed invention. Furthermore, the features of the dye distribution device 702 and the dyeing cartridge 703 can also be integrated with other embodiments described herein.

[0073] The dyeing box 703 also includes a dyeing surface 704 (e.g., a surface for receiving the arranged dyeing solution) and a trough 707. Although in Figures 7A-7D Not shown, but the dyeing box 703 may include a pair of lugs forming a lug structure. The lugs may be structurally and functionally similar to lugs 308, 408, 508, and 608 as described above.

[0074] According to one embodiment, for example, such as Figures 7A-7DAs shown, a dyeing solution (e.g., fixative, cleaning agent, etc.) is pre-deposited in one or more dye storage sections 709. According to operation 1002 of method 1000, the pre-deposited dye or other liquid is then deposited onto the dyeing surface 704 via high-pressure air jetting from an air jetting device 702A of the dye distribution device 702. According to various embodiments, deposition occurs when the dye distribution device 702 is coupled to one or more inlets 716 and the air jetting device 702A, and a measured amount of air pressure is discharged into one or more inlets 716 such that the liquid pre-deposited in one or more dye storage sections 709 is forced out of one or more ports 706. The distribution of dye or other liquids occurs with a surprisingly controlled degree during the dye or other liquid dispensing process. As described herein, the staining solution is deposited via a pressure jet of air from an air jet 702A, which ejects or otherwise disperses the liquid from the staining agent storage section 709 in the staining cartridge 703 without physical contact with the sample applied to the staining plate 301. In various embodiments, the air pressure can be adjusted to change the flow rate of the staining solution leaving the storage area. After the staining solution is deposited onto the staining surface 704, the staining solution can be distributed on the staining plate 301 according to operations 1004, 1006, and 1008.

[0075] Figures 7A-7D The illustration shows the application of multiple liquids (e.g., multiple dyeing solutions) onto a dyeing plate 301 using a dye distribution device 702. According to various embodiments, sequential distribution occurs when the dye distribution device 702 is sequentially coupled to each inlet 716 and an air injector 702A, and a measured amount of air pressure is discharged into the coupled inlet 716, forcing liquid pre-deposited in the corresponding dye storage section 709 out of the corresponding port 706. The dye distribution device 702 is not limited to, for example... Figures 7A-7D The tubular shape shown, and any suitable structure for dispersing the dye solution onto the dyeing surface (e.g., multiple dye distribution devices 702 capable of simultaneously distributing liquid pre-deposited within one or more dye storage portions 709) are within the scope of the claimed invention.

[0076] In a further embodiment, the dye storage section 709 may be configured to store dye powder. The dye powder may be hydrated to produce a dye solution before use in the dyeing process.

[0077] Figure 8A and Figure 8BA dyeing cartridge with a dye reservoir is shown. The dye reservoir 811 of the dyeing cartridge 803 can be combined with any dyeing cartridge and / or feature disclosed herein for automated dyeing. The dyeing cartridge 803 may include lugs 808, grooves 807, ports 806, discharge portions 805, dyeing surfaces 804, and raised structures 810. Each of these may be similar to those discussed above with respect to other embodiments.

[0078] In the staining cassette 803, the staining surface 804 further includes a staining reservoir 811. The staining reservoir 811 is a portion of the staining surface 804 located opposite the port 806, spaced apart from the larger principal portion of the staining surface 804. The staining reservoir 811 is characterized by a surface that slopes downwards or tapers as it extends away from the port 806 (e.g., relative to the plane defined by the principal portion of the staining surface 804). A first end of the staining reservoir 811 is disposed at the apex of the protrusion 810 where the port 806 is located. The staining reservoir 811 slopes downwards and extends away from the port 806 to a second end. The geometry of the staining reservoir 811 has the effect that when the substrate 301 (not depicted) is positioned to overlap the staining surface 804 and a chromosome accumulation is established, the staining reservoir 811 provides an overflow buffer for the chromosome accumulation. As the chromosome accumulation is established, the portion of the chromosome accumulation corresponding to the staining reservoir 811 has a gap that expands as it extends past the port 806.

[0079] In operation, the chromosome volume (including at least a portion of the staining reservoir) can be filled with staining solution by dispensing additional staining solution into the chromosome volume. When additional staining solution is added to the chromosome volume and the previous staining solution is removed, the staining reservoir 811 can act as a buffer in the event of a mismatch between the inflow and outflow rates, using the method discussed herein. Such a mismatch may occur due to, for example, subtle variations in the gaps, air bubbles in the fluid lines, and / or a potential mismatch in the pumping rates. If the inflow rate is too high compared to the outflow rate, the additional staining solution can fill the staining reservoir 811 without completely escaping the chromosome volume. If the outflow rate is too high compared to the inflow rate, the staining reservoir 811 can supply additional staining solution to port 806. In this way, the staining solution flowing into port 806 is not disturbed (e.g., due to the introduction of air or other ambient gases), which would otherwise disrupt capillary action within the chromosome volume and prevent normal flow of the staining solution.

[0080] In a further embodiment, a camera or other optical device can be trained on the staining reservoir 811 to measure the amount of liquid in the staining reservoir 811. Based on the amount of staining solution in the staining reservoir 811, the system can be configured to adjust the inflow and outflow rates of the staining solution to maintain an appropriate level of chromosome volume filling.

[0081] In a further embodiment, the staining cartridge may include an inlet port at a first end and an outlet port at a second end. A staining agent delivery device may be provided via the inlet port. As described herein, a first staining solution may be deposited on the staining surface through the inlet port and distributed on the staining plate by relative movement. A second staining solution may be deposited on the staining surface through the inlet port while simultaneously being withdrawn via the outlet port, resulting in the second staining solution being distributed on the staining plate.

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

[0083] As discussed above, air bubbles can be prevented from entering the chromosome volume by distributing the staining solution to areas or spaces leading to an unconstrained environment. However, air bubbles can present additional problems. As discussed above, the amount of staining solution delivered can be tightly controlled. Air bubbles present in the distribution line can prevent accurate measurement of the distribution amount because air bubbles represent undelivered liquid volume. In various embodiments, the presence of bubbles in the distribution line and / or delivered to the staining surface can be monitored by a camera or other imaging device or optical sensor. For example, an imaging device can monitor the distribution line. Air bubbles in the distribution line can be detected and their size estimated, and the amount of distributing volume adjusted accordingly. In another embodiment, an imaging device configured to monitor the flow from the staining agent delivery device to the staining surface can detect a lack of liquid flow indicating bubbles, and the system can then adjust the incoming flow rate and volume (e.g., by increasing the rate or flow rate) and the outgoing flow rate and volume (e.g., by pausing or slowing down) to ensure they remain balanced. In a further embodiment, the staining agent delivery system may include a mechanical bubble trap to prevent bubbles from interfering with the flow.

[0084] Combined with any of the methods and techniques described herein, control over the inflow and outflow of staining solutions with respect to chromosome volumes can be performed. The removal of the first staining solution and the introduction of the second staining solution can be performed by matching the volumetric flow rates of each staining solution. Matching the volumetric flow rates can include pumping out the first staining solution at an average volumetric flow rate substantially the same as that pumping in the second staining solution. Substantially the same average volumetric flow rate can include average flow rates that differ from each other by less than 5%, less than 3%, and less than 1% 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 provides at least two advantages. First, by ensuring that the outflow of the first staining solution is not greater than the inflow of the second staining solution, capillary action within the chromosome volume is not disrupted. If the outflow is too high, air may be drawn into the chromosome volume, which could disrupt capillary action at cavitation sites. Second, by ensuring that the inflow is not greater than the outflow, overflow of the chromosome volume can be prevented. If the inflow exceeds the outflow by too much, the chromosome volume may overflow, which could cause the meniscus edge of the chromosome volume to break.

[0085] In various embodiments, the volumetric flow rate of the staining solution can be matched by setting the inflow and outflow rates to be substantially the same—for example, by controlling the pumps driving the inflow and outflow. In further embodiments, the inflow and outflow volumetric flow rates can be monitored simultaneously, for example, by a flow meter or by one or more cameras or other optical devices configured to monitor chromosome volume and / or fluid lines (through which fluid is added to or removed from the chromosome volume). In various embodiments, the volumetric flow rate can be monitored by monitoring a combined flow rate—for example, a combination of input and output flow rates. For example, in embodiments where a camera or optical device monitors the chromosome volume, the camera can be configured to monitor the combined flow rate, for example, by monitoring the state of the chromosome volume (e.g., overfilled or underfilled), and the camera can monitor the combined input and output flow rates leading to the chromosome volume. The pumps driving the inflow and outflow of the staining solution can be controlled (e.g., by closed-loop control) based on information obtained from monitoring the volumetric flow rates. The closed-loop control can be performed by a processor and / or suitable hardware associated with the integrated sample staining and processing system 100. Closed-loop control can be advantageous in such systems for addressing minute variations in the process caused by factors such as slide misalignment, manufacturing variability, and air bubbles.

[0086] In various embodiments, the dyeing solution for performing the associated methods can be provided in various ways. For example, a dyeing cassette consistent with the embodiments discussed above can be configured to contain the dyeing solution. In other examples, the dyeing solution can be provided in a small, disposable container containing only enough dyeing solution to dye a single substrate. In a further example, a dyeing solution kit can be provided, packaging several different small, disposable dyeing solution containers together to perform a selected dyeing process.

[0087] In further embodiments, the staining cassette and associated system may include means or systems for increasing staining rate and reducing staining time. For example, one or more of the staining surface and staining plate (substrate) may be vibrated to reduce staining time. The staining surface and / or staining plate may be vibrated by an integrated sample staining and handling system and / or, as appropriate, by another means. In further embodiments, one or more of the staining surface and staining plate may be heated to reduce staining time. For example, the staining cassette disclosed herein may include an integrated heater, such as a resistance heater or other means. The staining plate disclosed herein may also be heated during the staining operation.

[0088] The staining apparatus and methods described herein can contribute to sample processing methods that provide increased accuracy and consistency in staining procedures, thereby reducing analysis time and increasing sample processing throughput. These advantages may be particularly valuable when provided in field settings to deliver rapid sample evaluation and thus improve results. The sample processing methods described herein are not limited to field processing and can be used to improve sample processing in any setting, including surgical tumor or tissue resection, remote or off-site pathology laboratories, clinical facilities, academic research institutions, etc.

[0089] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit 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 also be understood that the terms “comprising” and / or “including” as used in this specification specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0090] Other embodiments are described in the following terms: Clause 1. A method for distributing a staining solution on a substrate, the method comprising: arranging the substrate near 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 chromosome volume according to the overlap and the gap, the chromosome volume having one or more meniscus edges; distributing a portion of the staining solution on the staining surface; and distributing the staining solution on the substrate.

[0091] Clause 2. The method according to Clause 1, wherein arranging said portion of the staining solution comprises arranging said portion of the staining solution at the meniscus edge such that the staining solution is exposed to an unconstrained environment.

[0092] Clause 3. The method according to Clause 1, wherein distributing the dye solution includes increasing the overlap by relative movement of the substrate and the dyeing surface.

[0093] Clause 4. The method according to Clause 3, wherein the relative movement is a relative linear movement between the substrate and the dyed surface.

[0094] Clause 5. According to the method of Clause 3, wherein the portion of the staining solution is arranged at the meniscus edge at a first volume rate corresponding to the increase in chromosome volume caused by the increase in the overlap.

[0095] Clause 6. The method according to Clause 5, wherein the chromosome volume is filled with the staining solution without introducing air bubbles into the chromosome volume.

[0096] Clause 7. The method according to Clause 1 further comprises: depositing a second portion of the second staining solution at the meniscus edge of the chromosome volume on the staining surface at a second volume rate; and removing the portion of the staining solution from the chromosome volume at a third volume rate corresponding to the second volume rate.

[0097] Clause 8. The method according to Clause 7, wherein the deposition of the second portion and the removal of the portion are performed substantially simultaneously.

[0098] Clause 9. The method according to Clause 7, wherein said portion of said staining solution is removed from said chromosome volume via a port defined by a protrusion on said staining surface, wherein the size of said gap decreases toward said port.

[0099] Clause 10. The method according to Clause 9, wherein the removal of the portion of the staining solution via the port through the gap is performed without introducing air bubbles into the portion of the staining solution during the removal of the portion of the staining solution.

[0100] Clause 11. The method of claim 1, wherein the dyeing surface includes a dye reservoir extending away from the port, wherein the gap extends along the length of the dye reservoir extending away from the port.

[0101] Clause 12. The method according to Clause 11 further comprises: depositing a second portion of the second staining solution at a second volume rate at the meniscus edge of the chromosome accumulation on the staining surface; and removing the portion of the staining solution from the chromosome accumulation at a third volume rate corresponding to the second volume rate, wherein the staining reservoir is configured to provide a buffer for the mismatch between the second volume rate and the third volume rate.

[0102] Clause 13. The method according to Clause 9, wherein the port comprises a plurality of holes extending across at least 40% of the width of the substrate.

[0103] Clause 14. The method according to Clause 9, wherein the port includes a slit extending at least 40% of the width of the substrate.

[0104] Clause 15. The method according to Clause 7, wherein removing the portion of the staining solution from the chromosome volume at a third volume rate corresponding to the second volume rate comprises: monitoring a combined flow rate including the second volume rate and the third volume rate; and adjusting at least one of the second volume rate and the third volume rate based on the monitoring.

[0105] Clause 16. The method according to Clause 5, wherein distributing a portion of the staining solution at the meniscus edge at a first volume rate corresponding to the increase in chromosome volume caused by the increase in the overlap comprises: monitoring the overlap; and adjusting the first volume flow rate based on the monitoring.

[0106] Clause 17. The method according to Clause 1, wherein disposing of said portion of the dye solution includes releasing said portion of the dye solution from a dyeing cassette including the dyeing surface.

[0107] Clause 18. The method according to Clause 1, wherein said portion of the staining solution is a predetermined or measured portion.

[0108] Clause 19. The method according to Clause 1, wherein the stained surface is a coverslip.

[0109] Clause 20. The method according to Clause 1, wherein the substrate is a glass slide.

[0110] Clause 21. The method according to Clause 1, wherein the gap is a capillary gap.

[0111] Clause 22. The method according to Clause 1, wherein the height of the gap is substantially the same across the overlapping portion.

[0112] Clause 23. The method according to Clause 1, wherein the gap is defined by a gap profile of variable height.

[0113] Clause 24. The method according to Clause 1, wherein the gap is maintained by a lug structure.

[0114] Clause 25. The method according to Clause 1, wherein the gap is maintained by a robot actuator.

[0115] Clause 26. The method of Clause 1, wherein the distribution of the staining solution is performed by generating a corresponding movement between the staining surface and the substrate, the movement increasing the overlap between the staining surface and the substrate, the method further comprising, as the overlap is increased, filling the chromosome volume with a portion of the staining solution.

[0116] Clause 27. The method according to Clause 1, wherein the staining solution is a first staining solution, the method further comprising removing the portion of the first staining solution from the gap and adding a portion of a second staining solution.

[0117] Clause 28. The method according to Clause 27, wherein the removal and the addition are performed by pumping.

[0118] Clause 29. The method of Clause 27, wherein the removal and the addition are performed simultaneously.

[0119] Clause 30. The method of Clause 27, wherein the removal and the addition are performed sequentially.

[0120] Clause 31. The method according to Clause 27, wherein the removal and the addition are performed by corresponding movement between the stained surface and the substrate.

[0121] Clause 32. The method according to Clause 27, wherein at least one of the removal and the addition is performed by a combination of pumping and corresponding movement between the stained surface and the substrate.

[0122] Clause 33. The method according to Clause 1, wherein the substrate includes one or more protrusions, and the gap is defined between the stained surface and the one or more protrusions.

[0123] Clause 34. According to the method of Clause 1, the gap is a tapering gap that increases in height at the edge of the overlap.

[0124] Clause 35. The method according to Clause 34, wherein the gap tapers according to the raised structure on the dyed surface.

[0125] Clause 36. The method according to Clause 35, wherein the protruding structure has a volcano-like shape.

[0126] Clause 37. The method according to Clause 1, wherein the dyeing surface includes a port, and distributing the dye solution includes pumping the dye solution into the gap via the port.

[0127] Clause 38. The method according to Clause 37, wherein the pumping is performed with air gaps in the dyeing solution line.

[0128] Clause 39. The method according to Clause 1, wherein at least one of the substrate and the dyed surface includes a hydrophilic surface.

[0129] Clause 40. The method according to Clause 1, wherein at least one of the substrate and the dyed surface comprises a hydrophobic surface.

[0130] Clause 41. The method according to Clause 1 further includes hydrating the staining powder to produce the staining solution.

[0131] Clause 42. The method according to Clause 1, wherein the dyeing solution comprises at least one of a fixative, a color dye, and a water-based cleaning agent.

[0132] Clause 43. The method according to Clause 1, wherein distributing the dye solution on the substrate further comprises distributing a plurality of dye solutions on the substrate.

[0133] Clause 44. The method according to Clause 42, wherein distributing multiple dyeing solutions on the substrate includes distributing at least one fixative, at least two color dyes, and at least one water-based cleaning agent.

[0134] Clause 45. The method according to Clause 42, wherein the distribution of multiple dyeing solutions on the substrate includes a distribution fixative, a first color dye, a second color dye, and a water cleaning agent.

[0135] Clause 46. The method according to Clause 42, wherein it takes approximately 10 seconds to distribute each dye solution onto the substrate.

[0136] Clause 47. The method according to Clause 1, wherein the dye solution is distributed on the substrate in a time of less than 1 minute, less than 30 seconds, less than 10 seconds, less than 5 seconds, or less than 2 seconds.

[0137] Clause 48. The method according to Clause 1, wherein distributing the dye solution onto the substrate takes less than 1 second.

[0138] Clause 49. According to the method of Clause 9, the protrusion extends from the port by at least 2 mm.

[0139] Clause 50. The method according to any one of Clauses 1-49, wherein at least one of arranging the portion of the staining solution, arranging the staining surface, maintaining the gap, and distributing the staining solution is performed by an automated system.

[0140] Clause 51. An apparatus for staining a biological sample, the apparatus comprising: a staining structure including a base, a staining surface disposed on the base, and at least one gap maintaining structure disposed on the base; and a substrate configured to receive the biological sample; wherein: the substrate is configured to be arranged to establish an overlap between the staining surface and the substrate, the at least one gap maintaining structure is configured to maintain a gap between the staining surface and the substrate to define a chromosome volume according to the overlap and the gap, and the chromosome volume is configured to receive a staining solution and having one or more meniscus edges when the staining solution is received.

[0141] Clause 52. The apparatus according to Clause 51, wherein the dyeing cartridge is configured such that the dye solution is directed to an unconstrained environment at one or more meniscus edges.

[0142] Clause 53. The apparatus according to Clause 52, wherein the dyeing cassette is further configured to receive the dyeing solution at one or more meniscus edges.

[0143] Clause 54. The apparatus according to Clause 51, wherein the dyeing cassette is configured to distribute the dye solution on the substrate by increasing the overlap through relative movement of the substrate and the dyeing surface.

[0144] Clause 55. The apparatus according to Clause 54 further includes an actuator configured to increase the overlap by relative movement.

[0145] Clause 56. The apparatus according to Clause 51 further includes a port defined by a protrusion on the dyeing surface, wherein the size of the gap decreases toward the port, the port being configured to introduce the dye solution into the dyeing surface or remove the dye solution from the dyeing surface.

[0146] Clause 57. The apparatus according to Clause 56, wherein the dyeing surface includes a dyeing reservoir extending away from the port, wherein the gap extends along the length of the dyeing reservoir extending away from the port.

[0147] Clause 58. The apparatus according to Clause 57, wherein the port comprises a plurality of holes extending across at least 40% of the width of the substrate.

[0148] Clause 59. The apparatus according to Clause 56, wherein the port includes a slit extending at least 40% of the width of the substrate.

[0149] Clause 60. The apparatus according to Clause 51, wherein the dyeing cassette is configured to contain the dyeing solution.

[0150] Clause 61. The apparatus according to Clause 51, wherein the stained surface is a coverslip.

[0151] Clause 62. The apparatus according to Clause 51, wherein the substrate is a glass slide.

[0152] Clause 63. The apparatus according to Clause 51, wherein the gap is a capillary gap.

[0153] Clause 64. The apparatus according to Clause 51, wherein the height of the gap is substantially the same across the overlapping portion.

[0154] Clause 65. The apparatus according to Clause 51, wherein the gap is defined by a gap profile of variable height.

[0155] Clause 66. The apparatus pursuant to Clause 51 further includes a dye delivery device.

[0156] Clause 67. The apparatus according to Clause 51 further includes a discharge section configured to remove the dye solution.

[0157] Clause 68. The apparatus according to Clause 51, wherein the gap is a tapering gap that increases in height at the edge of the overlap.

[0158] Clause 69. The apparatus according to Clause 66, wherein the gap tapers according to the raised structure on the dyed surface.

[0159] Clause 70. The apparatus according to Clause 67, wherein the protruding structure has a volcano-like shape.

[0160] Clause 71. The apparatus according to Clause 51, wherein at least one of the substrate and the dyed surface comprises a hydrophilic surface.

[0161] Clause 72. The apparatus according to Clause 51, wherein at least one of the substrate and the dyed surface includes a hydrophobic surface.

[0162] Clause 73. The apparatus according to Clause 51, wherein the dyeing solution comprises at least one of a fixative, a color dye, and a water-based cleaning agent.

[0163] Clause 74. The apparatus according to Clause 51, wherein the gap maintaining structure includes one or more lugs having a top at a height above the dyeing surface and configured to support the substrate above the dyeing surface.

[0164] Clause 75. The apparatus according to Clause 51, wherein the gap maintaining structure includes one or more lugs having a top at a height below the dyeing surface and configured to support the substrate below the dyeing surface.

[0165] Clause 764. The apparatus according to Clause 74 further includes a groove disposed on the base and positioned between the dyed surface and the one or more lugs.

[0166] Clause 77. The apparatus according to Clause 51 further includes at least one additional dyed surface.

[0167] Clause 78. An apparatus for staining a biological sample, the apparatus comprising: a staining structure including a base, a staining surface disposed on the base, and at least one gap maintaining device; and a substrate configured to receive the biological sample; wherein: the substrate is configured to be arranged to establish an overlap between the staining surface and the substrate, the at least one gap maintaining device is configured to maintain a gap between the staining surface and the substrate to define a chromosome volume according to the overlap and the gap, and the chromosome volume is configured to receive a staining solution and having one or more meniscal edges when the staining solution is received.

[0168] Clause 79. The apparatus according to Clause 78, wherein the at least one gap maintaining device comprises a robot actuator.

[0169] The above embodiments are illustrative examples and should not be construed as limiting the invention to these specific embodiments. It should be understood that the various embodiments disclosed herein can be combined in combinations other than those specifically presented in the description and drawings. It should also be understood that, according to the examples, certain actions or events of any process or method described herein can be performed in different sequences, and can be added, combined, or omitted entirely (e.g., all described actions or events may not be necessary for implementing the method or process). Additionally, although for clarity, certain features of the various embodiments are described as being performed by a single module, device, or unit, it should be understood that the features and functions described herein can be performed by any combination of units or modules. Therefore, those skilled in the art can make various changes and modifications without departing from the spirit or scope of the invention as defined in the appended claims.

Claims

1. A method for distributing a dyeing solution on a substrate, the method comprising: The substrate is arranged near the dyeing surface to create an overlap between the dyeing surface and the substrate; Maintaining a gap between the stained surface and the substrate to define a chromosome volume according to the overlap and the gap, the chromosome volume having one or more meniscus edges; A portion of the dyeing solution is applied to the dyeing surface; and The dye solution is distributed on the substrate.

2. The method of claim 1, wherein arranging the portion of the staining solution comprises arranging the portion of the staining solution at one of the one or more meniscus edges, such that the staining solution is exposed to an unconstrained environment.

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

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

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

6. The method of claim 5, wherein the chromosome volume is filled with the staining solution without introducing air bubbles into the chromosome volume.

7. The method of claim 1, further comprising: A second portion of the second staining solution is deposited on the staining surface at a second volume rate at one of the meniscus edges of the chromosome accumulation; as well as The portion of the staining solution is removed from the chromosome volume at a third volume rate corresponding to the second volume 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 solution is removed from the chromosome volume via a port defined by a protrusion on the staining surface, wherein the size of the gap decreases toward the port.

10. The method of claim 9, wherein removing the portion of the staining solution through the gap via the port is performed without introducing air bubbles into the portion of the staining solution during removal.

11. The method of claim 1, wherein the dyeing surface includes a dye reservoir extending away from the port, wherein the gap extends along the length of the dye reservoir extending away from the port.

12. The method of claim 11, further comprising: A second portion of the second staining solution is deposited on the staining surface at a second volume rate at one of the meniscus edges of the chromosome accumulation; as well as The portion of the staining solution is removed from the chromosome volume at a third volume rate corresponding to the second volume rate. The staining reservoir is configured to provide a buffer for the mismatch between the second and third volume rates.

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

14. The method of claim 9, wherein the port comprises a slit extending at least 40% of the width of the substrate.

15. The method of claim 7, wherein removing the portion of the staining solution from the chromosome volume at a third volume rate corresponding to the second volume rate comprises: Monitoring includes a combined flow rate comprising the second volumetric rate and the third volumetric rate; as well as Adjust at least one of the second volume rate and the third volume rate based on the monitoring.

16. The method of claim 5, wherein distributing a portion of the staining solution at the meniscus edge at a first volumetric flow rate corresponding to the increase in chromosome volume caused by the increase in the overlap comprises: Monitor the overlapping portion; as well as The first volume flow rate is adjusted based on the monitoring.

17. The method of claim 1, wherein disposing of the portion of the dye solution comprises releasing the portion of the dye solution from a dyeing cassette including the dyeing surface.

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

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

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

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

22. The method of claim 1, wherein the heights of the gaps are substantially the same across the overlapping portion.

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 lug structure.

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

26. The method of claim 1, wherein the distribution of the dyeing solution is performed by generating a corresponding movement between the dyeing surface and the substrate, the movement increasing the overlap between the dyeing surface and the substrate. The method further includes filling the chromosome volume with a portion of the staining solution as the overlap is increased.

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

28. The method of claim 27, wherein the removal and the addition are performed by pumping.

29. The method of claim 27, wherein the removal and the addition are performed simultaneously.

30. The method of claim 27, wherein the removal and the addition are performed sequentially.

31. The method of claim 27, wherein the removal and the addition are performed by corresponding movement between the stained surface and the substrate.

32. The method of claim 27, wherein at least one of the removal and the addition is performed by a combination of pumping and corresponding movement between the stained surface and the substrate.

33. The method of claim 1, wherein the substrate includes one or more protrusions, and the gap is defined between the dyed surface and the one or more protrusions.

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

35. The method of claim 34, wherein the gap tapers according to the protrusion structure on the dyed surface.

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

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

38. The method of claim 37, wherein the pumping is performed with air gaps in the dyeing solution line.

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

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

41. The method of claim 1, further comprising hydrating the dyeing powder to produce the dyeing solution.

42. The method according to claim 1, wherein the dyeing solution comprises at least one of a fixative, a color dye, and a water-based cleaning agent.

43. The method of claim 1, wherein distributing the dye solution on the substrate further comprises distributing a plurality of dye solutions on the substrate.

44. The method of claim 42, wherein distributing the multiple dyeing solutions on the substrate comprises distributing at least one fixative, at least two color dyes, and at least one water-based cleaning agent.

45. The method of claim 42, wherein distributing the multiple dyeing solutions on the substrate comprises a distribution fixative, a first color dye, a second color dye, and a water cleaning agent.

46. ​​The method of claim 42, wherein distributing the multiple dye solutions on the substrate takes approximately 10 seconds to distribute each dye solution.

47. The method of claim 1, wherein distributing the dye solution 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.

48. The method of claim 1, wherein distributing the dye solution onto the substrate takes less than 1 second.

49. The method of claim 9, wherein the protrusion extends from the port by at least 2 mm.

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

51. An apparatus for staining biological samples, the apparatus comprising: A dyeing structure comprising a base, a dyeing surface disposed on the base, and at least one gap-maintaining structure disposed on the base; and A substrate configured to receive the biological sample; in: The substrate is configured to establish an overlap between the dyed surface and the substrate. The at least one gap-maintaining structure is configured to maintain the gap between the staining surface and the substrate, thereby defining the chromosome volume according to the overlap and the gap. The chromosome volume is configured to receive staining solution and has one or more meniscus edges when the staining solution is received.

52. The apparatus of claim 51, wherein the dyeing cassette is configured such that the dye solution is directed to an unconstrained environment at one or more meniscus edges.

53. The apparatus of claim 52, wherein the dyeing cassette is further configured to receive the dyeing solution at one or more meniscus edges.

54. The apparatus of claim 51, wherein the dyeing cassette is configured to distribute the dye solution on the substrate by increasing the overlap through relative movement of the substrate and the dyeing surface.

55. The apparatus of claim 54, further comprising an actuator configured to increase the overlap by relative movement.

56. The apparatus of claim 51, further comprising a port defined by a protrusion on the dyeing surface, wherein the size of the gap decreases toward the port, the port being configured to introduce the dye solution into the dyeing surface or remove the dye solution from the dyeing surface.

57. The apparatus of claim 56, wherein the dyeing surface includes a dye reservoir extending away from the port, wherein the gap extends along the length of the dye reservoir extending away from the port.

58. The apparatus of claim 57, wherein the port comprises a plurality of holes extending at least 40% of the width of the substrate.

59. The apparatus of claim 56, wherein the port comprises a slit extending at least 40% of the width of the substrate.

60. The apparatus of claim 51, wherein the staining cassette is configured to contain the staining solution.

61. The apparatus of claim 51, wherein the stained surface is a coverslip.

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

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

64. The apparatus of claim 51, wherein the height of the gaps is substantially the same across the overlapping portion.

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

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

67. The apparatus of claim 51, further comprising a discharge section configured to remove the staining solution.

68. The apparatus of claim 51, wherein the gap is a tapering gap that increases in height at the edge of the overlap.

69. The apparatus of claim 66, wherein the gap tapers according to the raised structure on the dyed surface.

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

71. The apparatus of claim 51, wherein at least one of the substrate and the dyed surface comprises a hydrophilic surface.

72. The apparatus of claim 51, wherein at least one of the substrate and the dyed surface comprises a hydrophobic surface.

73. The apparatus of claim 51, wherein the dyeing solution comprises at least one of a fixative, a color dye, and a water-based cleaning agent.

74. The apparatus of claim 51, wherein the gap maintaining structure includes one or more lugs having a top at a height above the dyeing surface and configured to support the substrate above the dyeing surface.

75. The apparatus of claim 51, wherein the gap maintaining structure includes one or more lugs having a top at a height below the dyeing surface and configured to support the substrate below the dyeing surface.

76. The apparatus of claim 74, further comprising a groove disposed on the base and positioned between the dyed surface and the one or more lugs.

77. The apparatus of claim 51, further comprising at least one additional dyed surface.

78. An apparatus for staining biological samples, the apparatus comprising: A dyeing structure comprising a base, a dyeing surface disposed on the base, and at least one gap maintaining device; and A substrate configured to receive the biological sample; in: The substrate is configured to establish an overlap between the dyed surface and the substrate. The at least one gap-maintaining device is configured to maintain a gap between the staining surface and the substrate to define a chromosome volume based on the overlap and the gap. The chromosome volume is configured to receive staining solution and has one or more meniscus edges when the staining solution is received.

79. The apparatus of claim 78, wherein the at least one gap maintaining device comprises a robot actuator.

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