Patent Application
20240033743
Cells within a tissue of a subject have differences in cell morphology and/or function due to varied analyte levels (e.g., gene and/or protein expression) within the different cells. The specific position of a cell within a tissue (e.g., the cell's position relative to neighboring cells or the cell's position relative to the tissue microenvironment) can affect, e.g., the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signaling and cross-talk with other cells in the tissue.
Spatial heterogeneity has been previously studied using techniques that only provide data for a small handful of analytes in the context of an intact tissue or a portion of a tissue, or provide a lot of analyte data for single cells, but fail to provide information regarding the position of the single cell in a parent biological sample (e.g., tissue sample).
Analytes within the biological sample are generally released through disruption (e.g., permeabilization) of the biological sample. Various methods of delivering permeabilization reagents to the biological sample are described herein.
All publications, patents, patent applications, and information available on the internet and mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.
Analytes within a biological sample are generally released through disruption (e.g., permeabilization) of the biological sample or other releasing means. Various methods of disrupting a biological sample are known, including permeabilization of the cell membrane of the biological sample. Described herein are methods of delivering a fluid to the biological sample, systems for sample analysis, and sample alignment methods. Also described herein are methods of delivering a fluid, including for example, a buffer or a permeabilization solutions having various detergents, buffers, proteases, and/or nucleases for different periods of time and at various temperatures.
In an aspect, a sample holder is provided. In one embodiment, the sample holder can include a first member including a first retaining mechanism configured to retain a first substrate comprising a sample. The first retaining mechanism can be configured to retain the first substrate disposed in a first plane. The sample holder can further include a second member including a second retaining mechanism configured to retain a second substrate including a reagent medium disposed in a second plane. The sample holder can further includes an alignment mechanism connected to one or both of the first member and the second member. The alignment mechanism can be configured to align the first and second members along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane. The adjustment mechanism may be configured to move the second member along the axis orthogonal to the second plane and/or move the first member along an axis orthogonal to the first plane. The first substrate and the second substrate may be positioned with a minimum spacing between the first substrate and the second substrate.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the sample holder may further include a spacing member positioned between the first member and the second member and configured to maintain the minimum spacing between the first member and the second member. The spacing member may be further configured to maintain an approximately parallel arrangement of the first substrate and second substrate. The alignment mechanism may include a pin extending from the second member. The alignment mechanism may include an aperture in the first member, the aperture sized and configured to mate with the pin. The sample holder may further include a coupling member coupled to the first substrate and the second substrate and configured to inhibit movement between the first substrate and the second substrate. The coupling member may include a magnet. The first member may be further configured to retain a third substrate disposed in the first plane. The third substrate may include a second sample.
The first retaining mechanism may include one or more spring members configured to apply a force to the first substrate to maintain contact between the first substrate and the first member. The second retaining mechanism may include one or more spring members configured to apply a force to the second substrate to maintain contact between the second substrate and the second member. The adjustment mechanism may be coupled to the second member and may include a linear actuator configured to move the second member along the axis orthogonal to the second plane. The linear actuator may be configured to move the second member along the axis orthogonal to the second plane at a velocity of at least 0.1 mm/sec. The linear actuator may be configured to move the second member along the axis orthogonal to the second plane with an amount of force of at least 0.1 lbs. The second member may include a heater configured to heat the first substrate and/or the second substrate. The second member may further include an insulation gasket coupled to the heater. The first retaining mechanism may be configured to retain the first substrate in a fixed position with respect to the first plane.
In another aspect, a sample holder is provided. In an embodiment, the sample holder may include a first member including a first retaining mechanism configured to retain a first substrate comprising a sample. The first retaining mechanism may be configured to retain the first substrate disposed in a first plane. The sample may be disposed in a first region of the first substrate. The sample holder may also include a second member including a second retaining mechanism configured to retain a second substrate including a reagent medium disposed in a second plane and disposed in a second region of the second substrate. The sample holder may also include an alignment mechanism connected to one or both of the first member and the second member, and configured to optically align the first region with the second region along the first plane and/or the second plane such that the sample contacts at least a portion of the reagent medium when the first region and the second region are aligned and when the first substrate and the second substrate are separated by a threshold distance. The sample holder may further include an adjustment mechanism configured to move the second member along the axis orthogonal to the second plane. The sample holder may also include an image capture device configured to capture, responsive to the alignment and during a time when the sample is in contact with at least a portion of the reagent medium, an image of the first region and the second region.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the sample holder may include a spacing member positioned between the first member and the second member and configured to provide a minimum spacing between the first substrate and the second substrate. The spacing member may be further configured to maintain an approximately parallel arrangement of the first substrate and the second substrate. The image capture device may be positioned inferior to the second member. The image capture device may be disposed on a track configured to move the image capture device along the track. The first member may be further configured to retain a third substrate disposed in the first plane, the third substrate comprising a second sample disposed in a third region of the third substrate. The second substrate may include the reagent medium further disposed in a fourth region of the second substrate. The alignment mechanism may be further configured to optically align the third region with the fourth region along the first plane and/or the second plane such that the second sample contacts at least a portion of the reagent medium when the third region and the fourth region are aligned and when the third substrate and the second substrate are separated by the threshold distance. The alignment mechanism may be further configured to optically align, responsive to optically aligning the first region with the second region, the third region with the fourth region along the first plane and/or the second plane. The alignment mechanism may include a sensor configure to determine a threshold alignment of the first region with the second region and a threshold alignment of the third region with the fourth region. The image capture device may be further configured to capture, responsive to the alignment of the third region with the fourth region and during a time when the second sample is in contact with at least a portion of the reagent medium, an image of the third region and the fourth region. The sample holder may further include a controller configured to control the alignment mechanism, the adjustment mechanism, and/or the image capture device. The controller may be configured to communicate with a user interface, the user interface configured to display information about the sample holder. The controller may be configured to receive a user input from the user interface. The controller may be configured to control the alignment mechanism, the adjustment mechanism, and/or the image capture device responsive to the user input. The sample holder may further include a sensor, wherein the controller is further configured to control, based on the sensor, the alignment mechanism, the adjustment mechanism, and/or the image capture device. The first retaining mechanism may be configured to retain the first substrate in a fixed position with respect to the first plane. The second member may further include a spring mechanism configured to couple to at least a portion of an inferior surface of the second substrate. The second member may further include a base configured to couple to an inferior surface of the second substrate and retain the second substrate on the second member. The spring mechanism may extend in a superior direction from the base. The spring mechanism may be configured to dispose the second substrate along the second plane at an angle different than the base. The first member may be configured to dispose the first substrate along the first plane at an angle parallel with the base. The adjustment mechanism may be configured to move the second member toward the first member such that the first substrate contacts at least a portion of the reagent medium. The reagent medium may be disposed in proximity and superior to the spring mechanism. The first substrate contacting the at least a portion of the reagent medium may urge the reagent medium in an opposite direction such that the reagent medium is displaced over the second substrate and/or the first substrate as the second member moves toward the first member and the second substrate and the first substrate become substantially parallel. The adjustment mechanism may be configured to move the second member toward the first member such that the spring mechanism compresses so that the first substrate, the second substrate, and the base are substantially parallel. The sample holder may further include a sensor configured to detect a bubble in the reagent medium responsive to the sample contacting at least a portion of the reagent medium.
In another aspect, a method of capturing an analyte from a biological sample disposed in a first region of a first substrate is provided. In an embodiment, the method may include mounting the first substrate on a first member of a support device, the first substrate disposed in a first plane, the first member configured to retain the first substrate in a fixed position with respect to the first plane. The method can further include mounting a second substrate on a second member of the support device. The second substrate can be disposed in a second plane and comprising a second region including a plurality of second capture probes. A second capture probe of the plurality of second capture probes can comprises a spatial barcode and a second capture domain. The method can further include aligning, along the first plane and/or the second plane, the first region with the second region such that the first region and the second region can be vertically aligned when the first substrate is positioned superior to the second substrate. The method can further include applying a reagent medium to the first substrate and/or the second substrate. The reagent medium can provide a permeabilization buffer between the biological sample and the second substrate. The method can further include positioning, responsive to the aligning and the applying, the second substrate such that the biological sample contacts at least a portion of the reagent medium when the first and second members are aligned and within a threshold distance along an axis orthogonal to the second plane allowing the analyte to migrate from the biological sample to the second substrate. The analyte can bind to the second capture domain.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the method may further include capturing, responsive to the positioning and during a time when the biological sample is in contact with at least a portion of the reagent medium, an image of the first region and the second region. The positioning may include compressing the reagent medium between the first substrate and the second substrate by moving the second member toward the first member to a minimum separation distance maintained by a spacing member positioned between the first substrate and the second substrate.
In another aspect, a method for aligning a sample area with an array area is provided. In an embodiment, the method may include receiving a first substrate within a first retaining mechanism of a sample holder. The first substrate may include a sample and the sample area. The method may also include receiving a second substrate within a second retaining mechanism of the sample holder. The second substrate may include an array, wherein the second substrate or the sample holder comprises an array area indicator associated with the array area of the second substrate. The method may also include adjusting a location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with the array area.
In another aspect, a method for aligning a sample area with an array area is provided. In an embodiment, the method may include receiving a first substrate within a first retaining mechanism of a sample holder. The first retaining mechanism comprises an array area indicator associated with the array area. The method may also include receiving a second substrate within a second retaining mechanism of the sample holder. The second substrate comprising an array, wherein the second substrate comprising a sample and the sample area. The method may further include adjusting a location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with the array area.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the first substrate may include a sample area indicator associated with the sample area of the first substrate and the adjusting further causes the sample area indicator to be aligned with respect to the array area indicator. The method may also include applying the sample area indicator to a first side of the first substrate prior to applying the sample to the first side of the first substrate. The sample area indicator may include a drawing applied to the first substrate by a user. The method may also include applying the sample area indicator to a second side of the first substrate after applying the sample to the first side of the first substrate. The sample area indicator may include a stamp or a sticker. Applying the sample area indicator to the second side of the first substrate may include drawing the sample area indicator with a marker by a user. The first substrate includes a fiducial mark.
The method can further include receiving, by a data processor of a computing device communicatively coupled to the sample holder, an image of the sample. The method can also include providing, by the data processor, the image of the sample for display via a display of the computing device. The method can further include receiving, by the data processor, an input identifying the sample area indicator based on the provided image. The received input may be provided to the computing device by a user, or by a remote computing device communicatively coupled to the computing device. The method may also include automatically determining, by the data processor, the sample area indicator, based on the image.
Adjusting the location of the first substrate relative to the second substrate can include viewing the first substrate and the second substrate within the sample holder. Adjusting the location of the first substrate relative to the second substrate can also include adjusting the first retaining mechanism and/or the second retaining mechanism to cause all or the portion of the sample area to be aligned with the array area. The array area indicator may be provided on the sample holder. The second substrate may be fixed in place within the sample holder and the first retaining mechanism is adjusted to cause all or the portion of the sample area to be aligned with the array area. The array area location indicator may be provided on a transparent surface of the second retaining mechanism. The array area location indicator may be provided on a first surface of the second retaining mechanism. The first surface may be opposite a second surface of the second retaining mechanism at which the second substrate is received.
The method may further include receiving, by a first data processor of a first computing device communicatively coupled to the sample holder, a plurality of video images acquired via a microscope coupled to the first computing device. The plurality of video images displaying the second substrate may be overlaid atop the first substrate within the sample holder. The method may also include providing, by the data processor, the plurality of video images for display via a display of the first computing device. The method may further include adjusting the first retaining mechanism to cause the sample area to be aligned with the array area. The method may also include providing the plurality of video images to a second data processor of a second computing device remote from and communicatively coupled to the first computing device. The second computing device configured to provide the plurality of video images for display. The second computing device may be further configured to receive an input from a user identifying the sample area indicator. The data processor of the second computing device may be further configured to control adjusting the location of the first substrate and/or the second substrate to cause the sample area to be aligned with the array area via a controller coupled to the sample holder and to the first computing device.
The method may also include receiving, by a first data processor of a first computing device communicatively coupled to the sample holder, a plurality of video images acquired via a microscope coupled to the computing device. The plurality of video images displaying the second substrate overlaid atop the first substrate within the sample holder. The method may further include automatically determining, by the first data processor, a sample area indicator on the first substrate based on the plurality of video images. The method may also include performing the adjusting automatically, by the first data processor, based on the automatically determined sample area indicator. Performing the aligning automatically may further include determining, by the first data processor, an area of the sample relative to an area of the array. Performing the aligning automatically may also include automatically determining a sample area indicator on the first substrate responsive to determining the area of the sample is less than the area of the array. Performing the aligning automatically may also include providing, by the first data processor, the sample area indicator as an outline of the sample. Performing the aligning automatically may further include performing the adjusting automatically, by the first data processor, based on the outline of the sample.
Performing the adjusting automatically may further include determining, by the first data processor, a fiducial mark located on the first substrate. Performing the adjusting automatically may also include performing the adjusting automatically, by the first data processor, based on the determined fiducial mark.
The method may also include receiving, by the data processor of the first computing device, an image of a sample and a sample area indicator from a second computing device communicatively coupled to the first computing device. The second computing device configured to acquire and provide the image of the sample and the sample area indicator to the data processor of the first computing device. The method may further include registering, by the data processor of the first computing device, the received image of the sample and the sample area indicator with at least one video image of the plurality of video images. The method may also include providing, by the data processor of the first computing device and based on the registering, a registered sample image via a display of the first computing device. The method may further include receiving an input to the first computing device identifying the sample area indicator based on the registered sample image. The method may also include performing the adjusting automatically based on the received input identifying the sample area indicator.
The array may be configured to capture analytes from the sample. The array area indicator may be located on the second substrate. The second substrate may include a reagent medium, and all or the portion of the sample area are aligned with the array area without the reagent medium contacting the sample.
In another aspect, a system for aligning a sample area with an array area is provided. In an embodiment, the system may include a sample holder. The sample holder may include a first retaining mechanism configured to retain a first substrate received within the first retaining mechanism. The first substrate comprising a sample and the sample area. The sample holder may also include a second retaining mechanism configured to retain a second substrate received within the second retaining mechanism. The second substrate may include an array, wherein the second substrate or the sample holder comprises an array area indicator associated with the array area of the second substrate or the sample holder. The sample holder may be configured to adjust a location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with the array area. The sample holder may also include an image capture device (e.g., microscope) operatively coupled to the sample holder and configured to view the first substrate and the second substrate within the sample holder. The sample holder may also include a first computing device communicatively coupled to the image capture device (e.g., microscope) and to the sample holder. The computing device may include a display, a first data processor, and a non-transitory computer readable storage medium storing computer readable and executable instructions, which when executed cause the first data processor to adjust the location of the first substrate relative to the second substrate to cause all or the portion of the sample area to be aligned with the array area.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the first substrate may include a sample area indicator associated with the sample area of the first substrate and the adjusting further causes the sample area indicator to be aligned with respect to the array area indicator. The sample area indicator may be applied to a first side of the first substrate prior to applying the sample to the first side of the first substrate. The sample area indicator may include a drawing applied to the first substrate by a user. The sample area indicator may be applied to a second side of the first substrate after applying the sample to the first side of the first substrate. The sample area indicator may include a stamp or a sticker. Applying the sample area indicator to the second side of the first substrate may include drawing the sample area indicator with a marker by a user. The first substrate may include a fiducial mark. The instructions may further cause the first data processor to perform operations including receiving an image of the sample acquired via the microscope. The instructions may further cause the first data processor to perform operations including providing the image of the sample for display via the display of the first computing device. The instructions may further cause the first data processor to perform operations including receiving an input identifying the sample area indicator based on the provided image.
The received input may be provided to the first computing device by a user, or by a remote computing device communicatively coupled to the first computing device. The instructions may further cause the first data processor to perform operations including automatically determining the sample area indicator, based on the image. Adjusting the location of the first substrate relative to the second substrate may include viewing the first substrate and the second substrate within the sample holder via the microscope. Adjusting the location of the first substrate relative to the second substrate may also include adjusting the first retaining mechanism and/or the second retaining mechanism to cause all or the portion of the sample area to be aligned with the array area. The array area indicator may be provided on the sample holder. The second substrate may be fixed in place within the sample holder and the first retaining mechanism is adjusted to cause all or the portion of the sample area to be aligned with the array area. The array area location indicator may be provided on a transparent surface of the second retaining mechanism. The array area location indicator may be provided on a first surface of the second retaining mechanism, the first surface opposite a second surface of the second retaining mechanism at which the second substrate is received.
The instructions may further cause the first data processor to perform operations including receiving a plurality of video images acquired via the microscope, the plurality of video images displaying the second substrate overlaid atop the first substrate within the sample holder. The instructions may further cause the first data processor to perform operations including providing the plurality of video images for display via the display of the first computing device. The instructions may further cause the first data processor to adjust the location of the first substrate relative to the second substrate by adjusting the first retaining mechanism to cause the sample area to be aligned with the array area location. The instructions may further cause the first data processor to provide the plurality of video images to a second data processor of a second computing device remote from and communicatively coupled to the first computing device. The second computing device may be configured to provide the plurality of video images for display.
The second computing device may be further configured to receive an input from a user identifying the sample area indicator. The second data processor may be further configured to control adjusting the location of the first substrate and/or the second substrate to cause the sample area to be aligned with the array area via a controller coupled to the sample holder and to the first computing device. The instructions may further cause the first data processor to perform operations including receiving a plurality of video images acquired via the image capture device (e.g., microscope), the plurality of video images displaying the second substrate overlaid atop the first substrate within the sample holder. The instructions may further cause the first data processor to perform operations including automatically determining a sample area indicator on the first substrate based on the plurality of video images. The instructions may further cause the first data processor to perform operations including automatically adjusting the location of the first substrate relative to the second substrate to cause all or the portion of the sample area to be aligned with the array area based on the automatically determined sample area indicator.
Automatically adjusting the location of the first substrate relative to the second substrate may also include automatically determining a sample area on the first substrate indicator responsive to determining the sample area of the first substrate is smaller than the array area of the second substrate. Automatically adjusting the location of the first substrate relative to the second substrate may also include providing the sample area indicator as an outline of the sample via the display. Automatically adjusting the location of the first substrate relative to the second substrate may also include performing the adjusting automatically based on the outline of the sample. Automatically adjusting the location of the first substrate relative to the second substrate may also include determining a fiducial mark located on the first substrate. Automatically adjusting the location of the first substrate relative to the second substrate may also include performing the adjusting automatically based on the determined fiducial mark.
The instructions may further cause the first data processor to perform operations including receiving an image of the sample and the sample area indicator from a second computing device communicatively coupled to the first computing device. The second computing device configured to acquire and provide the image of the sample and the sample area indicator to the first data processor of the first computing device. The instructions may further cause the first data processor to perform operations including registering the received image of the sample and the sample area indicator with at least one video image of the plurality of video images. The instructions may further cause the first data processor to perform operations including providing a registered sample image based on the registering, the registered sample image provided via the display of the first computing device. The instructions may further cause the first data processor to perform operations including receiving an input to the first computing device identifying the sample area indicator within the registered sample image. The instructions may further cause the first data processor to perform operations including performing the adjusting automatically based on the identified sample area indicator.
The array may be configured to capture analytes from the sample. The array area indicator may be located on the second substrate. The second substrate may include a reagent medium, and all or the portion of the sample area are aligned with the array area without the reagent medium contacting the sample
Also provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. In an embodiment, the method can include delivering a fluid to the area on the first substrate. A virtual gasket can surround the area on the first substrate and can contain the fluid within the area. The method can also include assembling the second substrate with the first substrate, thereby delivering the fluid to the array and the biological sample.
Also provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. The method can include delivering a fluid to the area on the second substrate. A virtual gasket can surround the area on the second substrate and contains the fluid on the array. The method can also include assembling the first substrate with the second substrate, thereby delivering the fluid to the array and the biological sample.
In some embodiments, the first substrate, the second substrate, or both can include a glass surface. In some embodiments delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate, the glass surface can be a glass slide.
In some embodiments, the first substrate and the second substrate can be axially aligned. In some embodiments, the first substrate and the second substrate can be in a cross configuration. In some embodiments, the virtual gasket may include a hydrophobic coating.
In another aspect, a method for delivering fluid to a biological sample disposed on a first substrate and an array disposed on a second substrate are provided. In embodiment, the method may include delivering the fluid to the first substrate and/or the second substrate, wherein at least one of the first substrate and the second substrate comprising a spacer. The method may also include assembling, subsequent to the delivering, a chamber including the first substrate, the second substrate, the biological sample, and the spacer. The spacer may be disposed between the first substrate and second substrate and may be configured to maintain the fluid within the chamber and maintain a separation distance between the first substrate and the second substrate. The spacer may be positioned to at least partially surround an area on the first substrate on which the biological sample may be disposed and/or the array disposed on the second substrate. The area of the first substrate, the spacer, and the second substrate may at least partially encloses a volume comprising the biological sample.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the chamber may include a partially or fully sealed chamber. The second substrate may include a hydrophobic area positioned away from a region of interest. The hydrophobic area may be configured to remove bubbles in the fluid from the chamber. The region of interest may include an area where the biological sample and the array overlap. The hydrophobic area may include a hydrophobic coating. The separation distance may include a distance of at least 2 μm. The separation distance may include a distance between about 5 μm to 25 μm. The second substrate may include the spacer. The first substrate may include the spacer. The method may further include, prior to delivering the fluid, applying the hydrophilic coating to the first substrate and/or the second substrate. The fluid may include a wetting agent. The fluid may include one or more permeabilization reagent(s). The spacer may include an air permeable spacer portion configured to vent a bubble from the fluid.
The method may include generating vibration to the first substrate and/or the second substrate. Delivering the fluid to the first substrate and/or the second substrate may include adjusting a humidity of the chamber. Delivering the fluid to the first substrate and/or the second substrate may include generating a vacuum to a region proximate to the first substrate and/or the second substrate. Delivering the fluid to the first substrate and/or the second substrate may include delivering the fluid to a region of the second substrate, the spacer comprising three sides partially surrounding the fluid. Delivering the fluid to the first substrate and/or the second substrate may include delivering the fluid to a region of the second substrate, the region outside an enclosed area of the second substrate, the enclosed area formed by the spacer. Delivering the fluid to the first substrate and/or the second substrate may include delivering the fluid to a region of the spacer, the region outside an enclosed area of the second substrate, the enclosed area formed by the spacer.
Assembling the chamber may include positioning, responsive to the delivering, the first substrate at an angle such that a dropped side of the first substrate contacts at least a portion of the fluid when the first substrate and the second substrate are within a threshold distance along an axis orthogonal to the second substrate. The dropped side may urge the fluid toward the three sides partially surrounding the fluid. The chamber may include a hydrophobic pattern at least partially surrounding the fluid and positioned to at least partially surround the area on the first substrate and/or the array disposed on the second substrate. The spacer may be formed from a uniform thickness material. The spacer may be formed from a material with a variable thickness. The spacer may be printed on the first substrate and/or the second substrate. The spacer may include a photoresist pattern. The spacer may include a beveled edge on one or more sides of the spacer.
In another aspect, a system is provided. In an embodiment, the system may include a first substrate comprising a coating for adhering a biological sample. The system may also include a second substrate comprising an array. The system may further include a spacer disposed between the first substrate and second substrate and configured to maintain a fluid within a chamber comprising the first substrate, the second substrate, the biological sample, and the spacer. The spacer may be further configured to maintain a separation distance between the first substrate and the second substrate. The spacer may be positioned to at least partially surround the biological sample on the first substrate and/or the array disposed on the second substrate.
In another aspect, a kit is provided. In an embodiment, the kit may include a second substrate comprising an array. The second substrate may further include a spacer surrounding the array. In another embodiment, a kit is provided. In an embodiment, the kit may include a first substrate comprising a biological sample. The first substrate may further include a spacer surrounding the biological sample. In another embodiment, a kit is provided. In an embodiment, the kit may include a spacer configured to be disposed between a first substrate and second substrate, and instructions for use according to a method of any one of the preceding claims.
In another aspect, a system for aligning a sample area with an array area is provided. In an embodiment, the system may include a sample holder. The sample holder may include a first retaining mechanism configured to retain a first substrate received within the first retaining mechanism, the first substrate comprising a sample and the sample area. The sample holder may also include a second retaining mechanism configured to retain a second substrate received within the second retaining mechanism. The second substrate may include an array, wherein the second substrate or the sample holder comprises an array area indicator associated with the array area of the second substrate or the sample holder. The sample holder may be configured to adjust a location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with the array area. The sample holder may further include at least one image capture device operatively coupled to the sample holder and configured to view the first substrate and the second substrate within the sample holder. The sample holder may also include a first computing device communicatively coupled to the at least one image capture device and to the sample holder. The computing device may include a display, a first data processor, and a non-transitory computer readable storage medium storing computer readable and executable instructions, which when executed cause the first data processor to adjust the location of the first substrate relative to the second substrate to cause all or the portion of the sample area to be aligned with the array area.
In some variations, one or more features disclosed herein including the following features may optionally be included in any feasible combination. For example, the first retaining mechanism may include a first set of alignment marks associated with a first portion of the sample area. The first retaining mechanism may include a second set of alignment marks associated with a second portion of the sample area. The second portion of the sample area may be smaller than the first portion of the sample area. The first retaining mechanism may include a third set of alignment marks indicating an exclusion zone on the first substrate. The sample holder may include a first member and a second member. The first member or the second member can be independently articulable. The first member and the second member may articulate to provide a uniform separation distance between the first substrate and the second substrate. The at least one image capture device may be configured inferior to the second member. The sample holder may include a plurality of image capture devices. The sample holder may include an LED illumination source. The sample holder may include a spring between the first member and the second member. The sample holder may include a sensor configured to determine a home position of a top portion of the sample holder relative to bottom portion of the sample holder as the top portion is closed upon the bottom portion. The sensor may include an optical beam-break sensor or an impedance sensor. The at least one image capture device may include a focus motor configured to actuate the at least one image capture device. The focus motor may include a cam configured to actuate the at least one image capture device in a horizontal manner.
In some embodiments, (i) the first retaining mechanism further comprises a trough, optionally wherein the trough extends around an area of the retaining mechanism onto which the first substrate is received, or (ii) the second retaining mechanism further comprises a trough, optionally wherein the trough extends around an area of the retaining mechanism onto which the second substrate is received, or (iii) the first retaining mechanism further comprises a first trough and the second retaining mechanism further comprises a second trough, optionally wherein the first trough extends around an area of the retaining mechanism onto which the first substrate is received, optionally wherein the second trough extends around an area of the retaining mechanism onto which the second substrate is received. In some embodiments, the system or sample holder further comprises a hinge resistance mechanism that provides resistance upon closure of the first member and the second member of the sample holder. In some embodiments, the system or sample holder can include a closure feedback mechanism configured in a top portion of the sample holder. The closure feedback mechanism can be received within a receiving portion of a bottom portion of the sample holder when the top portion is brought into contact with the bottom portion. In some embodiments, the receiving portion can include a slot to receive the closure feedback mechanism. In some embodiments, the closure feedback mechanism includes a hole that can be received within and engage the receiving portion. In some embodiments, the closure feedback mechanism can be secured to the top portion of the sample holder via a pin. In some embodiments, a gasket is configured on the second retaining mechanism of the sample holder.
Additionally, various methods of delivering fluids (e.g., a permeabilization solution) to a biological sample are described herein including the use of a substrate holder. In some examples, a gasket can be disposed between a first substrate including a biological sample and a second substrate including an array (e.g., a spatial array). In some examples, the gasket may not include apertures. In some embodiments, the gasket can include one or more apertures. In some embodiments, the one or more apertures can include a hydrophobic coating. In some examples, fluid can be delivered to a partially sealed chamber through an aperture in the gasket. In some examples, the fluid can be delivered via capillary flow. In some examples, the fluid can be delivered with a syringe. In some embodiments, the method can include patterning the hydrophobic coating surrounds one or more regions of interest in the biological sample.
Also provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate. In an embodiment, the method can include assembling a partially sealed chamber comprising the first substrate, the second substrate, the biological sample, and a gasket. The gasket can be disposed between the first substrate and second substrate, and can surround the area on the first substrate and/or the array disposed on the second substrate. The area of the first substrate, the gasket, and the second substrate can at least partially enclose a volume comprising the biological sample and delivering the fluid to the partially sealed chamber through one or more apertures of the gasket, can thereby delivering the fluid to the array and the biological sample.
In some embodiments, the hydrophobic coating can be applied with a stamp. In some embodiments, the hydrophobic coating can be a paraffin-based wax. In some embodiments, the hydrophobic coating can cover the all, or a portion of, the first substrate outside the area on the first substrate. In some embodiments, the hydrophobic coating can cover all, or a portion of, the second substrate outside the array on the second substrate. In some embodiments, the hydrophobic coating can be applied in a pattern. In some embodiments, patterning the hydrophobic coating surrounds one or more regions of interest in the biological sample. In some embodiments delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate, the hydrophobic coating can include electrowetting.
In some embodiments, delivering the fluid can include delivering the fluid to the first substrate. In some embodiments, delivering the fluid can include delivering the fluid to the second substrate. In some embodiments, the method can include, prior to assembling, providing the biological sample on the area of the first substrate and the array disposed on the second substrate. In some embodiments, delivering the fluid can be at temperature of about 5° C. to about 80° C.
In some embodiments, the method can include permeabilizing the biological sample. In some embodiments, the second substrate can include one or more dried permeabilization reagent(s) disposed thereon. In some embodiments, the fluid can solubilize the one or more dried permeabilization reagent(s). In some embodiments, the fluid can include one or more permeabilization reagent(s). In some embodiments, the permeabilizing can be performed for about 1 minute to about 90 minutes. In some embodiments, the permeabilizing can include heating the first substrate, the second substrate, or both the first substrate and the second substrate. In some embodiments, the permeabilizing of the biological sample can be controlled by modulating the temperature of the fluid. In some embodiments, modulating the temperature of the fluid can include heating to at least about 40° C.
In some embodiments, the one or more permeabilization reagent(s) can be selected from the group consisting of: a detergent, an enzyme, and a buffer. In some embodiments, the one or more dried permeabilization reagent(s) can include one or both of a detergent and an enzyme. In some embodiments, the detergent can include one or more of SDS, N-lauroylsarcosine, saponin, or any combination thereof. In some embodiments, the enzyme can include one or more of proteinase K, pepsin, collagenase, trypsin, or any combination thereof. In some embodiments, the buffer can include TE, TAE, TBE, and PBS. In some embodiments, delivering the one or more permeabilization reagent(s) can include delivering the detergent, the enzyme, and the buffer sequentially in any order. In some embodiments, delivering the one or more permeabilization reagent(s) can include delivering the detergent, the enzyme, and the buffer as a mixture. In some embodiments, delivering can include delivering the one or more permeabilization reagent(s) two or more times to the partially sealed chamber.
In some embodiments, the method can further includes imaging the biological sample. In some embodiments, the biological sample can be a tissue section. In some embodiments, the biological sample can be a fresh-frozen tissue section. In some embodiments, the biological sample can be a fixed biological sample. In some embodiments, the fixed biological sample can be a formalin-fixed paraffin-embedded biological sample.
In some embodiments, the array can include a plurality of features. In some embodiments, the array can include about 5,000 features. In some embodiments, a feature of the plurality of features can be selected from the group consisting of a bead, a spot, an inkjet spot, a well, a hydrogel pad, and a nanoparticle. In some embodiments, a plurality of capture probes can be attached to the bead. In some embodiments, a capture probe of the plurality of capture probes can include a capture domain, and a spatial barcode unique to the feature. In some embodiments, the capture domain of the capture probe can include a poly(T) sequence. In some embodiments, the capture probe can include one or more of: a functional domain, a cleavage domain, a unique molecular identifier, or any combination thereof.
Also provided herein are kits including a first substrate comprising a coating for adhering a biological sample, a second substrate comprising an array, and a gasket. In some kits, the gasket comprises no apertures. In some embodiments, the gasket can include one aperture for fluid delivery. In some kits, the gasket can include two apertures for fluid delivery. In some kits, the first substrate can include one or more via-holes for fluid delivery. In some kits, the second substrate can include one or more via-holes for fluid delivery. In some kits, the kit can include a paraffin-based crayon. In some kits, the kit can include a hydrophobic coating stamp. In some kits, the kit can include a reverse transcriptase and a nuclease. In some kits, the kit can include one or more permeabilization agent(s). In some kits, the first substrate, the second substrate, and the gasket can be assembled into a partially sealed chamber. In some kits, the kit can include instructions for assembling a partially sealed chamber.
Where values are described in terms of ranges, it should be understood that the description includes the disclosure of all possible sub-ranges within such ranges, as well as specific numerical values that fall within such ranges irrespective of whether a specific numerical value or specific sub-range is expressly stated.
The term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection, unless expressly stated otherwise, or unless the context of the usage clearly indicates otherwise.
Various embodiments of the features of this disclosure are described herein. However, it should be understood that such embodiments are provided merely by way of example, and numerous variations, changes, and substitutions can occur to those skilled in the art without departing from the scope of this disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of this disclosure.
The following drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner. Like reference symbols in the drawings indicate like elements.
This disclosure describes apparatus, systems, methods, and compositions for spatial analysis of biological samples. This section describes certain general terminology, analytes, sample types, and preparative steps that are referred to in later sections of the disclosure. For example, the terms and phrases: spatial analysis, barcode, nucleic acid, nucleotide, probe, target, oligonucleotide, polynucleotide, subject, genome, adaptor, adapter, tag, hybridizing, hybridize, annealing, anneal, primer, primer extension, proximity ligation, nucleic acid extension, polymerase chain reaction (PCR) amplification, antibody, affinity group, label, detectable label, optical label, template switching oligonucleotide, splint oligonucleotide, analytes, biological samples, general spatial array-based analytical methodology, spatial analysis methods, immunohistochemistry and immunofluorescence, capture probes, substrates, arrays, analyte capture, partitioning, analysis of captured analytes, quality control, multiplexing, and/or the like are described in more detail in PCT Patent Application Publication No. WO2020/123320, the entire contents of which are incorporated herein by reference.
Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from single-cell or multicellular organisms (e.g., a mammal). The relationship between cells and their relative locations within a tissue sample may be critical to understanding disease pathology. Spatial transcriptomics technology may allow scientists to measure all the gene activity in a tissue sample and map where the activity is occurring. This technology and embodiments described herein may lead to new discoveries that may prove instrumental in helping scientists gain a better understanding of biological processes and disease.
Tissues and cells obtained from a mammal, e.g., a human, often have varied analyte levels (e.g., gene and/or protein expression) which can result in differences in cell morphology and/or function. The position of a cell or a subset of cells (e.g., neighboring cells and/or non-neighboring cells) within a tissue can affect, e.g., the cell's fate, behavior, morphology, and signaling and cross-talk with other cells in the tissue. Information regarding the differences in analyte levels (gene and/or protein expression) within different cells in a tissue of a mammal can also help physicians select or administer a treatment that will be effective and can allow researchers to identify and elucidate differences in cell morphology and/or cell function in the single-cell or multicellular organisms (e.g., a mammal) based on the detected differences in analyte levels within different cells in the tissue. Differences in analyte levels within different cells in a tissue of a mammal can also provide information on how tissues (e.g., healthy and diseased tissues) function and/or develop. Differences in analyte levels within different cells in a tissue of a mammal can also provide information of different mechanisms of disease pathogenesis in a tissue and mechanism of action of a therapeutic treatment within a tissue.
The spatial analysis methodologies herein provide for the detection of differences in an analyte level (e.g., gene and/or protein expression) within different cells in a tissue of a mammal or within a single cell from a mammal. For example, spatial analysis methodologies can be used to detect the differences in analyte levels (e.g., gene and/or protein expression) within different cells in histological slide samples, the data from which can be reassembled to generate a three-dimensional map of analyte levels (e.g., gene and/or protein expression) of a tissue sample obtained from a mammal, e.g., with a degree of spatial resolution (e.g., single-cell resolution).
Spatial heterogeneity in developing systems has typically been studied via RNA hybridization, immunohistochemistry, fluorescent reporters, or purification or induction of pre-defined subpopulations and subsequent genomic profiling (e.g., RNA-seq). Such approaches, however, rely on a relatively small set of pre-defined markers, therefore introducing selection bias that limits discovery. These prior approaches also rely on a priori knowledge. RNA assays traditionally relied on staining for a limited number of RNA species. In contrast, single-cell RNA-sequencing allows for deep profiling of cellular gene expression (including non-coding RNA), but the established methods separate cells from their native spatial context.
Spatial analysis methodologies described herein provide a vast amount of analyte level and/or expression data for a variety of multiple analytes within a sample at high spatial resolution, e.g., while retaining the native spatial context.
The binding of an analyte to a capture probe can be detected using a number of different methods, e.g., nucleic acid sequencing, fluorophore detection, nucleic acid amplification, detection of nucleic acid ligation, and/or detection of nucleic acid cleavage products. In some examples, the detection is used to associate a specific spatial barcode with a specific analyte produced by and/or present in a cell (e.g., a mammalian cell).
Capture probes can be, e.g., attached to a surface, e.g., a solid array, a bead, or a coverslip. In some examples, capture probes are not attached to a surface. In some examples, capture probes can be encapsulated within, embedded within, or layered on a surface of a permeable composition (e.g., any of the substrates described herein).
Non-limiting aspects of spatial analysis methodologies are described in WO 2011/127099, WO 2014/210233, WO 2014/210225, WO 2016/162309, WO 2018/091676, WO 2012/140224, WO 2014/060483, U.S. Pat. Nos. 10,002,316, 9,727,810, U.S. Patent Application Publication No. 2017/0016053, Rodrigues et al., Science 363(6434):1463-1467, 2019; WO 2018/045186, Lee et al., Nat. Protoc. 10(3):442-458, 2015; WO 2016/007839, WO 2018/045181, WO 2014/163886, Trejo et al., PLoS ONE 14(2):e0212031, 2019, U.S. Patent Application Publication No. 2018/0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol. 15:50, 2017, WO 2017/144338, WO 2018/107054, WO 2017/222453, WO 2019/068880, WO 2011/094669, U.S. Pat. Nos. 7,709,198, 8,604,182, 8,951,726, 9,783,841, 10,041,949, WO 2016/057552, WO 2017/147483, WO 2018/022809, WO 2016/166128, WO 2017/027367, WO 2017/027368, WO 2018/136856, WO 2019/075091, U.S. Pat. No. 10,059,990, WO 2018/057999, WO 2015/161173, and Gupta et al., Nature Biotechnol. 36:1197-1202, 2018, the entire contents of which are incorporated herein by reference and can be used herein in any combination. Further non-limiting aspects of spatial analysis methodologies are described herein.
Embodiments described herein may map the spatial gene expression of complex tissue samples (e.g., on tissue slides) with slides (e.g., gene expression slides) that utilize analyte and/or mRNA transcript capture and spatial barcoding technology for library preparation. A tissue (e.g., fresh-frozen, formalin-fixed paraffin-embedded (FFPE), or the like) may be sectioned and placed in proximity to a slide with thousands of barcoded spots, each containing millions of capture oligonucleotides with spatial barcodes unique to that spot. Once tissue sections are fixed, stained, and permeabilized, they release mRNA which binds to capture oligos from a proximal location on the tissue. A reverse transcription reaction may occur while the tissue is still in place, generating a cDNA library that incorporates the spatial barcodes and preserves spatial information. Barcoded cDNA libraries are mapped back to a specific spot on a capture area of the barcoded spots. This gene expression data may be subsequently layered over a high-resolution microscope image of the tissue section, making it possible to visualize the expression of any mRNA, or combination of mRNAs, within the morphology of the tissue in a spatially-resolved manner.
At 105, the capture probes can be optionally cleaved from the array, and the captured analytes can be spatially-barcoded by performing a reverse transcriptase first strand cDNA reaction. A first strand cDNA reaction can be optionally performed using template switching oligonucleotides. At 106, the first strand cDNA can be amplified (e.g., using polymerase chain reaction (PCR)), where the forward and reverse primers flank the spatial barcode and analyte regions of interest, generating a library associated with a particular spatial barcode. In some embodiments, the cDNA comprises a sequencing by synthesis (SBS) primer sequence. The library amplicons may be sequenced and analyzed to decode spatial information.
Embodiments described herein relating to preparing the biological sample on the slide may beneficially allow a user to confirm pathology or relevant regions on a tissue section, to confirm selection of best or undamaged tissue sections for analysis, to improve array-tissue alignment by allowing placement anywhere on the pathology slide. Further, workflows for preparing the biological sample on the slide may empower user or scientists to choose what to sequence (e.g., what tissue section(s) to sequence).
In some embodiments, the extension reaction can be performed separately from the sample handling apparatus described herein that is configured to perform the exemplary sandwiching process 104.
The sandwich configuration of the sample 302, the pathology slide 303 and the slide 304 may provide advantages over other methods of spatial analysis and/or analyte capture. For example, the sandwich configuration may reduce a burden of users to develop in house tissue sectioning and/or tissue mounting expertise. Further, the sandwich configuration may decouple sample preparation/tissue imaging from the barcoded array (e.g., spatially-barcoded capture probes 306) and enable selection of a particular region of interest of analysis (e.g., for a tissue section larger than the barcoded array). The sandwich configuration also beneficially enables spatial transcriptomics assays without having to place a tissue section 302 directly on the gene expression slide (e.g., slide 304) which may reduce cost and risk of mistakes/issues during sample preparation. The sandwich configuration may also provide an improvement of sensitivity and spatial resolution by vertically confining target molecules within the diffusion distance.
The methods described above for analyzing biological samples, such as the sandwich configuration described above, can be implemented using a variety of hardware components. In this section, examples of such components are described. However, it should be understood that in general, the various steps and techniques discussed herein can be performed using a variety of different devices and system components, not all of which are expressly set forth.
In some aspects, the velocity of the moving plate (e.g., closing the sandwich) may affect bubble generation or trapping within the permeabilization solution 305. In some embodiments, the closing speed is selected to minimize bubble generation or trapping within the permeabilization solution 305. In some embodiments, the closing speed is selected to reduce the time it takes the flow front of a reagent medium from an initial point of contact with the first and second substrate to sweep across the sandwich area (also referred to herein as “closing time”, see, e.g.,
In some aspects, when the sample handling apparatus 1400 is in an open position (as in
In some aspects, after the first member 1404 closes over the second member 1410, an adjustment mechanism (not shown) of the sample handling apparatus 1400 may actuate the first member 1404 and/or the second member 1410 to form the sandwich configuration for the permeabilization step (e.g., bringing the first substrate 1406 and the second substrate 1412 closer to each other and within a threshold distance for the sandwich configuration). The adjustment mechanism may be configured to control a speed, an angle, or the like of the sandwich configuration.
In some embodiments, the tissue sample (e.g., sample 302) may be aligned within the first member 1404 (e.g., via the first retaining mechanism 1408) prior to closing the first member 1404 such that a desired region of interest of the sample 302 is aligned with the bar-coded array of the gene expression slide (e.g., the slide 304), e.g., when the first and the second substrates are aligned in the sandwich configuration. Such alignment may be accomplished manually (e.g., by a user) or automatically (e.g., via an automated alignment mechanism). After or before alignment, spacers may be applied to the first substrate 1406 and/or the second substrate 1412 to maintain a minimum spacing between the first substrate 1406 and the second substrate 1412 during sandwiching. In some aspects, the permeabilization solution (e.g., permeabilization solution 305) may be applied to the first substrate 1406 and/or the second substrate 1412. The first member 1404 may then close over the second member 1410 and form the sandwich configuration. Analytes and/or mRNA transcripts 308 May be captured by the capture probes 306 and may be processed for spatial analysis.
In some embodiments, during the permeabilization step, the image capture device 1420 may capture images of the overlap area (e.g., overlap area 710) between the tissue 302 and the capture probes 306. If more than one first substrates 1406 and/or second substrates 1412 are present within the sample handling apparatus 1400, the image capture device 1420 may be configured to capture one or more images of one or more overlap areas 710.
Analytes within a biological sample are generally released through disruption (e.g., permeabilization, digestion, etc.) of the biological sample or may be released without disruption. Various methods of permeabilizing (e.g., any of the permeabilization reagents and/or conditions described herein) a biological sample are described herein, including for example including the use of various detergents, buffers, proteases, and/or nucleases for different periods of time and at various temperatures. Additionally, various methods of delivering fluids (e.g., a buffer, a permeabilization solution) to a biological sample are described herein including the use of a substrate holder (e.g., sandwich assembly, sandwich configuration, as described herein)
Provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate.
In some embodiments and with reference to
In some aspects, it may be possible to reduce or eliminate bubble formation between the slides using a variety of filling methods and/or closing methods. For example, during the sandwiching of the two slides (e.g., the pathology slide 303 and the slide 304) it may be possible to provide an angled closure of the slides to suppress or eliminate bubble formation.
Workflows described herein include contacting a drop of the liquid reagent disposed on a first substrate (e.g., the first substrate 406, 1406, or the like) or a second substrate (e.g., the second substrate 412, 1412, or the like) with at least a portion of a first substrate (e.g., the first substrate 406, 1406, or the like) or second substrate (e.g., the second substrate 412, 1412, or the like), respectively. In some embodiments, the contacting comprises bringing the two substrates into proximity such that the sample on the first substrate is aligned with the barcode array of capture probes on the second substrate. In some instances, the contacting is achieved by arranging the first substrate and the second substrate in an angled sandwich assembly as described herein.
As shown in
As shown in
At step 1810, the drop side of the angled slide 1706 contacts the drop 1705 first. The contact of the slide 1706 with the drop 1705 may form a linear or low curvature flow front that fills uniformly with the slides closed.
At step 1815, the slide 1706 is further lowered toward the slide 1712 (or the slide 1712 is raised up toward the slide 1706) and the dropped side of the slide 1706 may contact and may urge the liquid reagent toward the side opposite the dropped side and creating a linear or low curvature flow front that may prevent or reduce bubble trapping between the slides. As further shown, the spring 1715 may begin to compress as the slide 1706 is lowered.
At step 1820, the drop 1705 of liquid reagent fills the gap (e.g., the gap 307) between the slide 1706 and the slide 1712. The linear flow front of the liquid reagent may form by squeezing the drop 1705 volume along the contact side of the slide 1712 and/or the slide 1706. Additionally, capillary flow may also contribute to filling the gap area. As further shown in step 1820, the spring 1715 may be fully compressed such that the slide 1706, the slide 1712, and the base 1704 are substantially parallel to each other.
In some aspects, the angled closure of
As shown in
In some aspects, the angled closure of
In some embodiments, the drop (e.g., drop 1705) includes permeabilization reagents (e.g., any of the permeabilization reagents described herein). In some embodiments, the rate of permeabilization of the biological sample is modulated by delivering the permeabilization reagents (e.g., a fluid containing permeabilization reagents) at various temperatures.
In some embodiments, the permeabilization reagents are dried permeabilization reagents. In some embodiments, the dried permeabilization reagents are disposed on a substrate (e.g., the first substrate, the second substrate). In some embodiments, delivering the fluid (e.g., by any of the fluid delivery methods described herein) solubilizes the dried permeabilization reagents. In some embodiments, solubilizing the permeabilization reagents results in permeabilization of the biological sample. In some embodiments, delivering the fluid to solubilize dried reagents is delivered via an aperture in a gasket. In some embodiments, delivering the fluid to solubilize dried reagents is delivered through a via-hole. In some embodiments, the fluid solubilizing dried reagents includes the use of a syringe. In some embodiments, the fluid solubilizing dried reagents includes the capillary flow.
Spatial analysis workflows generally involve contacting a sample with an array of features. Aligning a sample with a reagent medium (or, in some embodiments, the array) is an important step in performing spatialomic (e.g., spatial transcriptomic) assays. The ability to efficiently generate robust experimental data for a given sample can depend greatly on the alignment of the sample and the reagent medium (or the array). Traditional techniques require samples to be placed directly onto a reagent medium (or the array). For example, current methods of aligning biological samples with barcoded areas in spatial transcriptomics assays involve a user carefully placing the biological sample onto a substrate that includes a plurality of barcoded probes. This approach can require skilled personnel and additional experimental time to prepare a section of the sample and to mount the section of the sample directly on the reagent medium (or the array). Misalignment of the sample and the reagent medium (or the array) can result in wasted reagent medium (or a wasted array), extended sample preparation time, and inefficient use of samples, which may be limited in quantity.
The systems, methods, and computer readable mediums described herein can enable efficient and precise alignment of samples and arrays, thus facilitating the spatial transcriptomic imaging and analysis workflows or assays described herein. Thus, in some embodiments, an advantage of the devices described is providing an alignment tool for users to align a sample with a barcoded area. Samples, such as portions of tissue, can be placed on a first substrate. The first substrate can include a slide onto which a user can place a sample of the tissue. An array, (e.g., such as a reagent array, or such as a spatially barcoded array) can be formed on a second substrate. The second substrate can include a slide and the array can be formed on the second substrate. The use of separate substrates for the sample and the array can beneficially allow user to perform the spatialomic (e.g., spatial transcriptomic) assays described herein without requiring the sample to be placed onto an array substrate. The sample holder and methods of use described herein can improve the ease by which users provide samples for spatialomic (e.g., spatial transcriptomic) analysis. For example, the systems and methods described herein alleviate users from possessing advanced sample or tissue sectioning or mounting expertise. Additional benefits of utilizing separate substrates for samples and arrays can include improved sample preparation and sample imaging times, greater ability to perform region of interest (ROI) selection, and more efficient use of samples and array substrates. The devices of the disclosure can reduce user error during the assay analysis, thereby also reducing sample analysis costs. In some embodiments, another advantage of the devices of the disclosure is a reduction in the number of aberrations or imaging imperfections that may arise due to user error in aligning a biological sample with a barcoded area of the substrate. In some embodiments, the devices of the disclosure allow for pre-screening of samples for areas of interest. In some embodiments, the devices of the disclosure allow for archived samples to be examined.
The sample substrate and the array substrate, and thus, the sample and the array, can be aligned using the instrument and processes described herein. The alignment techniques and methods described herein can generate more accurate spatialomic (e.g., spatial transcriptomic) assay results due to the improved alignment of samples with an array (e.g., such as a reagent array, or such as a spatially barcoded array).
In some embodiments, a workflow described herein comprises contacting a sample disposed on an area of a first substrate with at least one feature array of a second substrate. In some embodiments, the contacting comprises bringing the two substrates into proximity such that the sample on the first substrate may be aligned with the barcoded array on the second substrate. In some instances, the contacting is achieved by arranging the first substrate and the second substrate in a sandwich assembly. In some embodiments, the workflow comprises a prior step of mounting the sample onto the first substrate.
Alignment of the sample on the first substrate with the array on the second substrate may be achieved manually or automatically (e.g., via a motorized alignment). In some aspects, manual alignment may be done with minimal optical or mechanical assistance and may result in limited precision when aligning a desired region of interest for the sample and the barcoded array. Additionally, adjustments to alignment done manually may be time-consuming due to the relatively small time requirements during the permeabilization step.
It may be desirable to perform real-time alignment of a tissue slide (e.g., the pathology slide 303) with an array slide (e.g., the slide 304 with barcoded capture probes 306). In some implementations, such real-time alignment may be achieved via motorized stages and actuators of a sample handling apparatus (e.g., the sample handling apparatus 400, the sample handling apparatus 1400, or the like).
An exemplary spatial analysis workflow disclosed herein is provided. In some instances, the methods include providing a first substrate that includes a biological sample and a second substrate that includes a plurality of capture probes. In some instances, the plurality of capture probes include oligonucleotide probes. In some instances, the plurality of capture probes includes analyte capture agents that can detect an analyte of interest (e.g., a protein) as described herein. In some instances, the plurality of capture probes includes analyte capture agents that can detect an analyte of interest (e.g., a protein) as described herein. In some instances, the plurality of capture probes includes a capture domain comprising a sequence complementary to a capture handle sequence present in an analyte capture agent. The methods can be performed in an order determined by a person skilled in the art. For example, as an exemplary overview, a first substrate include a biological sample. The biological sample then is stained using any of the methods described herein. In some instances, the biological sample is imaged, capturing the stain pattern created during the stain step. In some instances, the biological sample then is destained. After destaining, in some instances, a second substrate that includes capture probes as described herein is added to the first substrate. In some instances, the biological sample is permeabilized using methods disclosed herein (e.g., a solution that includes proteinase K and SDS). Permeabilization releases the analytes from the biological sample. Analytes then migrate from the first substrate and are captured by the second substrate. In some instances, after capture, the analytes and/or the probe can be amplified and the sequence can be determined using methods disclosed herein.
In some instances, the first substrate and the second substrate are arranged in a sandwich assembly, e.g., as described herein. It is noted that the terms first substrate and second substrate do not necessarily connote the particular order or location of the biological sample or capture probes. For example, in one instance, the first substrate includes the biological sample and the second substrate includes capture probes. In another instance, the first substrate includes capture probes and the second substrate includes the biological sample. In some embodiments, the tissue permeabilization process begins when the sample is contacted with the permeabilization buffer. During the permeabilization process, analytes are released from the sample. In some embodiments, analytes that are released from the permeabilized sample diffuse to the surface of the second substrate and are captured on the feature array (e.g., on barcoded probes). In some instances, there is a gap between the first and the second substrate. In some instances, the gap is about 1, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 12.5, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 μm or more. In some embodiments, second substrate is placed in direct contact with the sample on the first substrate ensuring no diffusive spatial resolution losses. In some embodiments, an alignment mechanism is configured to maintain a separation between the first and second substrates when the first and second substrates are aligned. In some embodiments, the alignment mechanism is configured to maintain the separation such that at least a portion of the sample on the first substrate contacts at least a portion of the reagent medium on the second substrate. In some embodiments, the separation between the first and second substrates is between 2 microns and 1 mm, measured in a direction orthogonal to a surface of the first substrate that supports the sample. In some instances, the first substrate and the second substrate are separated (e.g., pulled apart). In some embodiments, the sample analysis (e.g., cDNA synthesis) can be performed on the first substrate after the first substrate and the second substrate are separated. In some embodiments, the substrate comprising the biological sample can be discarded or archived after the first substrate and the second substrate are separated.
In some aspects, the movement of the first member 404A may be performed by an alignment mechanism configured to move the slide 303A (e.g., the first substrate 406, the first substrate 1406, the slide 1706, or the like) along a first plane (e.g., the xy plane of the slide 303A). In some implementations, the alignment mechanism may be configured to move the gene expression slide 304 (e.g., the second substrate 412, the second substrate 1412, the slide 1712, or the like) along a second plane (e.g., the xy plane of the slide 304).
In some aspects, the movement of the first member 404B may be performed by an alignment mechanism configured to move the slide 303B (e.g., the first substrate 406, the first substrate 1406, the slide 1706, or the like) along a first plane (e.g., the xy plane of the slide 303B). In some implementations, the alignment mechanism may be configured to move the gene expression slide 304 (e.g., the second substrate 412, the second substrate 1412, the slide 1712, or the like) along a second plane (e.g., the xy plane of the slide 304).
At 2320, a second substrate can be received within a second retaining mechanism of the sample handling apparatus 400. The second substrate can include an array of reagent medium formed within an array area indicator identifying the array on the second substrate. In some embodiments, the array area indicator can be provided on the sample handling apparatus 400. A user can provide or position the second substrate within the second retaining mechanism of the sample handling apparatus 400. The second retaining mechanism can include one or more spring members configured to apply a force to the second substrate to maintain contact between the second substrate and a second member of the sample holder on which the second retaining mechanism is configured.
At 2330, a location of the first substrate can be adjusted relative to the second substrate to cause all or a portion of the sample area of the first substrate to be aligned with the array area of the second substrate. In some embodiments, adjusting the location of the first substrate relative to the second substrate can be performed to cause the sample area indicator to be aligned with the array area indicator. In some embodiments, the location of the first substrate relative to the second substrate can be adjusted by a user. For example, the user can manually manipulate the first member and/or the second member of the sample holder so as to adjust a location of the first substrate and/or the second substrate within the sample holder to cause the sample area to be aligned with the array area. In some embodiments, the location of the first substrate can be adjusted relative to the second substrate, which can be fixed in position within the sample handling apparatus 400. In some embodiments, the location of the second substrate can be adjusted relative to the first substrate, which can be fixed in position within the sample handling apparatus 400. In some embodiments, the second substrate can be fixed in place within the sample handling apparatus 400 and the first retaining mechanism can be adjusted to cause all or a portion of the sample area to be aligned with the array area.
In some embodiments, a user can adjust the location of the first substrate and/or the second substrate while viewing the first substrate and/or the second substrate within the sample handling apparatus 400. For example, the user can view the first substrate and the second substrate via a microscope of the instrument configured to provide the sample holder within a field of view of the microscope. In some embodiments, the instrument can include a display providing a view of the first substrate and the second substrate within the sample handling apparatus.
In some embodiments, adjusting the location of the first substrate relative to the second substrate can further include viewing the first substrate and the second substrate within the sample holder and adjusting the first retaining mechanism and/or the second retaining mechanism to cause all or a portion of the sample area to be aligned with the array area. In this way, the sample handling apparatus 400 can advantageously support efficient and precise alignment by providing multiple, different ways to perform the alignment. In some embodiments, the adjusting can be performed in the absence of a sample area indicator configured on the first substrate and/or in the absence of an array area indicator configured on the second substrate.
In some embodiments, the location of the first substrate and/or the second substrate can be adjusted within the sample holder by a user interacting with a physical positioning device configured on the sample handling apparatus 400, or on the instrument while viewing the first substrate and the second substrate. The physical positioning device can include a joy stick, a pointing stick, a button, or the like. In some embodiments, the instrument can be configured with computer-readable, executable instructions stored in a memory of the instrument. The instructions, when executed, can perform the adjusting automatically based on image data associated with the sample handling apparatus 400, the first substrate, and/or the second substrate. In some embodiments, the instrument can be configured with a display providing a graphical user interface (GUI). A user can interact with the GUI to adjust the location of the first substrate relative to the second substrate to cause all or a portion of the sample area indicator to be aligned with respect to the array area indicator.
The sample handling apparatus 400 can be configured to enable adjustment of the first substrate 2405 and/or the second substrate 2425 along a first axis 2445 and a second axis 2450. The first axis 2445 can be considered a later axis within a transverse plane corresponding to the mounting surface in which the first substrate 2405 and the second substrate 2425 are received within the sample handling apparatus 400. The second axis 2450 can be considered a longitudinal axis within the transverse plane corresponding to the mounting surface in which the first substrate 2405 and the second substrate 2425 are received within the sample handling apparatus 400.
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At 2720, the data processor can provide the image of the sample for display via a display of the computing device. In some embodiments, the image of the sample can be provided for display via a GUI configured within the display of the computing device.
At 2730, the data processor can receive an input identifying the sample area indicator based on the provided image. For example, the display of the computing device can include a touch-screen display configured to receive a user input identifying the sample area indicator on the displayed image. In some embodiments, the GUI can be configured to receive a user provided input identifying the sample area indicator.
At 2740, the data processor can automatically determine the sample area indicator based on the image. The data processor can be configured to access and execute computer-readable, executable instructions configured to automatically determine the sample area indicator based on a variety of features included in the image. For example, the data processor can automatically determine the sample area indicator based on an outline of the tissue present within the image. This approach can be used when the sample area is smaller than the array area. In some embodiments, the data processor can automatically determine the sample area indicator based on a stamp or a sticker that is visible in the image and was applied to the first substrate by a user. In some embodiments, the data processor can automatically determine the sample area indicator based on a fiducial mark located on the first substrate that is visible in the image. In some embodiments, the data processor can automatically determine the sample area indicator based on a drawing that is visible in the image and was applied to the first substrate by a user.
In some embodiments, the data processor can access and execute computer-readable, executable instructions configured to automatically determine the sample area indicator based on sample area indicator data which can be stored in a memory of the computing device. In some embodiments, the sample area indicator data can be imported into the computing device from a second computing device that is remote from and communicatively coupled to the computing device automatically determining the sample area indicator associated with the sample in the image.
In some embodiments, the data processor can access and execute computer-readable, executable instructions configured to automatically determine the sample area indicator based on processing the sample image using image segmentation functionality. In some embodiments, the data processor can access and execute computer-readable, executable instructions configured to automatically determine the sample area indicator based on a type of sample, a size of sample, a shape of the sample, and/or an area of the sample.
At 2920, the data processor can provide the plurality of video images for display via a display of the computing device. In some embodiments, the plurality of video images can be provided for display via a GUI configured within the display of the computing device. In some embodiments, the plurality of video images can be provided to a data processor of a second computing device. The second computing device can be remote from the first computing device and can be communicatively coupled to the first computing device at which the plurality of video images were first received. The second computing device can be configured to provide the plurality of video images for display via a display of the second computing device. In some embodiments, the second computing device can be configured to receive an input from a user identifying a sample area indicator associated with the sample positioned on the first substrate. The user can provide the input identifying the sample area indicator to the second computing device as previously described above.
At 2930, a user can manually adjust a first retaining mechanism of the sample handling apparatus 400 to cause the sample area of the first substrate to be aligned with the array area of the second substrate. In some embodiments, the user can adjust the first retaining mechanism of the sample handling apparatus 400 to cause the sample area of the first substrate to be aligned with an array area configured within the sample handling apparatus 400. The user can adjust the first retaining mechanism based on viewing the plurality of video images provided by the first computing device or the second computing device.
At 2940, in addition, or in alternative, to the manual adjustment performed at 2930, the data processor of the first computing device can automatically determine the sample area indicator based on the plurality of video images. The data processor of the first computing device can be configured to access and execute computer-readable, executable instructions configured to automatically determine the sample area indicator based on a variety of features included in the plurality of video images. For example, the data processor can automatically determine the sample area indicator based on an outline of the tissue present within the plurality of video images. This approach can be used when the sample area is smaller than the array area. In some embodiments, the data processor can automatically determine the sample area indicator based on a stamp or a sticker that is visible in the plurality of video images and was applied to the first substrate by a user. In some embodiments, the data processor can automatically determine the sample area indicator based on a fiducial mark located on the first substrate that is visible in the plurality of video images. In some embodiments, the data processor can automatically determine the sample area indicator based on a drawing that is visible in the plurality of video images and was applied to the first substrate by a user.
In some embodiments, the data processor can access and execute computer-readable, executable instructions configured to automatically determine the sample area indicator based on sample area indicator data which can be stored in a memory of the computing device. In some embodiments, the sample area indicator data can be imported into the computing device from a second computing device that is remote from and communicatively coupled to the computing device automatically determining the sample area indicator associated with the sample in the plurality of video images.
At 2950, the data processor of the first computing device can perform the adjusting automatically based on the automatically determined sample area indicator. The computing device can be configured to automatically adjust the location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with an array area of the second substrate via a controller that can be communicatively coupled to the sample handling apparatus 400 and to the first computing device. The controller can receive input signals from the data processor and can generate control signals causing the first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 400 and there by adjust the location of the first substrate or the second substrate, respectively.
In some embodiments, the data processor of a second computing device, communicatively coupled to the data processor of the first computing device, can similarly be coupled to the controller and to the sample handling apparatus 400. The data processor of the second computing device can generate input signals to the controller and can cause the controller to generate control signals causing first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 400. In this way, the location of the first substrate and/or the second substrate can be controlled and adjusted such that the sample area of the first substrate can be aligned with the array area of the second substrate.
At 3020, the data processor can automatically determine a sample area indicator on the first substrate responsive to determining the area of the sample is less than the area of the array. For example, after the tissue slide is scanned and the outline of the tissue is determined using image processing, the outline may be compared to the area of the array to determine the area of the sample is less than the area of the array.
At 3030, the data processor can provide the sample area indicator as an outline of the sample. For example, the sample area indicator can be provided in a display of the computing device.
At 3040, the data processor can perform the adjusting automatically based on the outline of the sample. As described above, the data processor of the computing device can be configured to automatically adjust the location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with an array area of the second substrate via a controller that can be communicatively coupled to the sample handling apparatus 400 and to the computing device. For example, the data processor may be configured to fit the outline of the sample within the array area. The alignment of the outline may be to the array itself, a virtual outline on a UI, or some alignment reference marks elsewhere in the instrument (sample handling apparatus 400) that may indicate the array position. The controller can receive input signals from the data processor and can generate control signals causing the first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 400 and thereby adjust the location of the first substrate or the second substrate, respectively to fit the outline of the sample within the array area.
At 3120, the data processor can perform the adjusting automatically based on the determined fiducial mark. As described above, the data processor of the computing device can be configured to automatically adjust the location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with an array area of the second substrate via a controller that can be communicatively coupled to the sample handling apparatus 400 and to the computing device. In some aspects, the adjusting may be based on the location of the determined fiducial. For example, the fiducial may provide a reference point for aligning the first substrate with the second substrate. The controller can receive input signals from the data processor and can generate control signals causing the first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 400 and there by adjust the location of the first substrate or the second substrate, respectively.
At 3220, the data processor of the first computing device can register the received image of the sample and the sample area indicator with at least a video image of a plurality of video images. The plurality of video images can be acquired via an image capture device 2120, such as a microscope, a camera, an optical sensor, an imaging device, or the like, communicatively coupled to the data processor of the first computing device.
At 3230, the data processor of the first computing device can provide, based on the image registration, a registered sample image via a display of the first computing device. For example, the registered sample image can be provided in a display of the first computing device.
At 3240, an input identifying the sample area indicator in the registered sample image can be received at the first computing device. For example, a user can provide an input to a GUI provided in a display of the first computing device. In some embodiments, the display can receive the input directly from the user or via an input device, such as a mouse or a stylus, coupled to the display.
At 3250, the data processor can perform the adjusting automatically based on the received input identifying the sample area indicator. The computing device can be configured to automatically adjust the location of the first substrate relative to the second substrate to cause all or a portion of the sample area to be aligned with an array area of the second substrate via a controller that can be communicatively coupled to the sample handling apparatus 400 and to the first computing device. The controller can receive input signals from the data processor and can generate control signals causing the first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 400 and there by adjust the location of the first substrate or the second substrate, respectively.
After alignment of the slides 303 and 304 (e.g., as shown in
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After adding the permeabilization solution (e.g., permeabilization solution 305) to the aligned slides, it may be beneficial to capture images of the aligned tissue sample 302 and/or the barcoded capture probes 306 to aid in mapping gene expressions to locations of the tissue sample 302. As such, the image capture device 2120 may be configured to capture images of the aligned tissue sample 302, regions of interest 2202, and/or the barcoded capture probes 306 during a permeabilization step.
In some aspects, the permeabilization step may occur within one minute and it may be beneficial for the image capture device 2120 to move quickly between the different sandwiched slides and regions of interest. Although a single image capture device 2120 is shown, more than one image capture device 2120 may be implemented.
In some aspects, the sandwich may be opened by moving the second member 410 away from the first members 404, or vice versa. The opening may be performed by the adjustment mechanism 415 of the sample handling apparatus 400.
While workflows 2100, 2200, 3300, and 3400 are shown and described with respect to the sample handling apparatus 400, the workflows 2100, 2200, 3300, and 3400 may also be performed with respect to the sample handling apparatus 1400, the sample handling apparatus 3500, or another sample handling apparatus in accordance with the implementations described herein. In some embodiments, the processes 2300, 2700, 2900, 3000, 3100, and 3200 may also be performed with respect to the sample handling apparatus 1400, the sample handling apparatus 3500, or another sample handling apparatus in accordance with the implementations described herein.
As shown, the sample handling apparatus 3500 includes an adjustment mechanism 415, a linear guide 3516, a trans-illumination source 3517, one or more heaters 1108, first members 404A and 404B, tissue slides 303A and 303B, tissue samples 302A and 302B, a gene expression slide 304, and the image capture device 2120. In the example of
In the example sandwich maker workflows described herein, a liquid reagent (e.g., the permeabilization solution 305) may fill a gap (e.g., the gap 307) between a tissue slide (e.g., slide 303) and a capture slide (e.g., slide 304 with barcoded capture probes 306) to warrant or enable transfer of target molecules with spatial information. Described herein are examples of filling methods that may suppress bubble formation and suppress undesirable flow of transcripts and/or target molecules or analytes. Robust fluidics in the sandwich making described herein may preserve spatial information by reducing or preventing deflection of molecules as they move from the tissue slide to the capture slide.
The one or more spacers 6805 may be configured to maintain a separation distance between the first substrate and the second substrate. The one or more spacers 6805 can be placed on the first substrate adjacent to the biological sample 302 and in between the first substrate and the second substrate. The one or more spacers 6805 can be placed on the second substrate adjacent to the array 306 and in between the first substrate and the second substrate. By doing so, the one or more spacers 6805 can create a chamber (e.g., chamber 6810) in which solutions (e.g., a buffer, a permeabilization solution 305) are contained throughout the permeabilization and analyte migration process. In some embodiments, more than one spacer is used. In some embodiments, the one or more spacers 6805 have a height of about 2 μm, about 12.5 μm, about 15 μm, about 17.5 μm, about 20 μm, about 22.5 μm, or about 25 μm. In some embodiments, the height of each spacer has a height of about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1000 μm. The one or more spacers 6805 may be formed of a material having uniform thickness or of a material having a variable (e.g., beveled) thickness.
In some embodiments, the one or more spacers 6805 may create a fully or partially enclosed chamber around the biological sample (e.g., tissue sample 302 or a region of interest) and/or the array 306. The fully enclosed one or more spacers 6805 can be configured to any shape. In some embodiments, the fully enclosed (e.g., encompassed) chamber created by the one or more spacers 6805 is one of a square or a rectangle. In some embodiments, one or more spacers 6805 conform to the shape of the biological sample 302. For example, the one or more spacers 6805 are shown herein to have an example shape, an example height, and maintain an example separation distance (e.g., 12.5 μm), although other values and shapes are possible and may depend on the liquid reagent, the biological sample 302, the capture probes 306, or the like.
In some aspects, any combination of bubble venting or bubble removing features may be applied to the chamber, the first substrate, and/or the second substrate. For example, air permeable spacers (e.g., spacers 3610) may be configured to vent out trapped bubbles. Further, bubble venting holes disposed on the first substrate, the second substrate, and/or a spacer may be placed at strategic locations to vent bubbles. In some aspects, a sonication or vibration device may be configured to generate vibration on the first substrate and/or the second substrate during closing of the sandwich to reduce the chance of a bubble sticking to a surface of the first substrate or the second substrate. Additionally, it may be possible to increase a humidity of the chamber during sandwich closing to facilitate the filling process of the permeabilization solution or liquid reagent. Further, it may be possible to generate a vacuum in the chamber during closing to reduce or eliminate the chance of bubble trapping.
In some aspects, any combination of the one or more spacers 3610, the hydrophobic area 3720, or the like may be implemented to achieve flow and/or bubble suppression. In some embodiments, the one or more spacers 3610 and/or the hydrophobic area 3720 may be disposed on either the first substrate (e.g., the pathology slide 303) or the second substrate (e.g., the slide 304).
In some aspects, the alignment of the tissue sample 302 with the capture probes 306 shown in
Also provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate, including, delivering a fluid to the area on the first substrate, where a virtual gasket surrounds the area on the first substrate and contains the fluid within the area and assembling the second substrate with the first substrate, thereby delivering the fluid to the array and the biological sample.
Also provided herein are methods for delivering a fluid to a biological sample disposed on an area of a first substrate and an array disposed on a second substrate, including, delivering a fluid to the area on the second substrate, where a virtual gasket surrounds the area on the second substrate and contains the fluid on the array and assembling the first substrate with the second substrate, thereby delivering the fluid to the array and the biological sample.
In some embodiments, the biological sample is disposed on a first substrate. In some embodiments an array (e.g., a substrate including capture probes) is on a second substrate. In some embodiments, the first substrate including the biological sample and the second substrate including the array (e.g., a spatial array) are brought in proximity to one another such that the first substrate and the second substrate are disposed proximally to each other.
As used herein, a “partially sealed chamber” is a chamber between a first substrate and a second substrate, where a gasket is disposed between the first substrate and the second substrate.
In some embodiments of any of the methods for delivery a fluid described herein, the first substrate, the second substrate, or both, can be any of the substrates described herein. In some embodiments, the first substrate is a glass surface. In some embodiments, the second substrate is a glass surface. In some embodiments, the first substrate and the second substrate are both glass surfaces. In some embodiments, the glass surface is a glass slide. In some embodiments, the first substrate, the second substrate, or both are glass slides.
In some embodiments, the first substrate and the second substrate can be axially aligned. For example, the second substrate can be placed on top of the first substrate, or vice versa, in substantially the same orientation as the first substrate. In some embodiments, the first substrate and the second substrate are aligned in a cross-configuration. For example, the second substrate can be placed on top of the first substrate, or vice versa, at approximately a 90° angle to the first substrate. In some embodiments, the second substrate can be placed on top of the first substrate, or vice versa, at about a 40°, about 45°, about 50°, about 55°, about 60°, about 65°, about 70°, about 75°, about 80°, or about 85° angle relative to the first substrate.
In some embodiments, a gasket is disposed on the first substrate prior to aligning the first substrate and second substrate (e.g., axially, cross-configuration). In some embodiments, the gasket surrounds (e.g., encompasses) the biological sample. In some embodiments, a gasket is disposed on the second substrate prior to aligning the first substrate and the second substrate. In some embodiments, the gasket surrounds (e.g., encompasses) the array on the substrate. In some embodiments, the gasket has no apertures (e.g., an opening). In some embodiments, the gasket includes one or more apertures and a hydrophobic coating is disposed at one or more apertures in the gasket to help prevent overflow after delivery of the fluid (e.g., a permeabilization solution).
In some embodiments, the gasket can be made of rubber, silicone, or a similar material to create a seal with the first substrate. In some embodiments, the gasket can be made of a material that is hydrophobic. Accordingly, different fluids, including a permeabilization solution, can be delivered to the various apertures of the gasket. In some embodiments, the engagement of the bottom of the gasket and top of the gasket in contact with the first substrate and the second substrate creates ample pressure to maintain a partially sealed chamber where fluid (e.g., a buffer, a permeabilization solution) is delivered. In some embodiments, the gasket is a virtual gasket.
As used herein, a “virtual gasket” is a hydrophobic coating that functions similar to a physical gasket such that fluid delivered within the virtual gasket is contained within the perimeter of the virtual gasket. In some embodiments, the hydrophobic coating helps localize the fluid (e.g., permeabilization solution) over the biological sample, including a region of interest. In some embodiments, the hydrophobic coating controls the volume between the first substrate and the second substrate after alignment (e.g., axially, cross-configuration) assembly. In some embodiments, the hydrophobic coating is applied with a stamp. For example, the hydrophobic coating can be applied with a stamp to the first substrate, the second substrate, or both. In some embodiments, the virtual gasket is drawn. In some embodiments, the virtual gasket is drawn with a wax or a paraffin-based crayon. In some embodiments, the virtual gasket is patterned. For example, the hydrophobic coating can be applied in a pattern to the first substrate, the second substrate, or both. In some embodiments, the hydrophobic coating encompasses the biological sample, the array, or both. In some embodiments, the hydrophobic coating is applied in a similar pattern to the physical gaskets described herein. For example, the hydrophobic coating can have no apertures, one aperture, or two or more apertures. In some embodiments, the hydrophobic coating is extended beyond the encompassed biological sample, array, or both to prevent capillary flow between the first substrate and the second substrate. In some embodiments, the hydrophobic coating is applied patterned in a grid. For example, the hydrophobic coating can be applied (e.g., applied by any of the methods described herein) to encompass one or more biological samples, one or more arrays (e.g., spatial array), or both on a substrate. In some embodiments, the hydrophobic coating can be applied to encompass 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more biological samples, one or more arrays (e.g., spatial array), or both on a substrate (e.g., a first substrate, a second substrate). In some embodiments, the hydrophobic coating is patterning is controlled dynamically via electro wetting.
In some embodiments, a spacer is used to separate the two substrates (e.g., the first substrate and the second substrate). Spacers can be placed adjacent to the biological sample and in between the first substrate and the second substrate. By doing so, spacers can create a chamber in which solutions (e.g., a buffer, a permeabilization solution) are contained throughout the permeabilization and analyte migration process. In some embodiments, more than one spacer is used. In some embodiments, a spacer has a height of about 10 μm, about 12.5 μm, about 15 μm, about 17.5 μm, about 20 μm, about 22.5 μm, or about 25 μm. In some embodiments, the height of each spacer has a height of about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 400 μm, about 500 μm, about 600 μm, about 700 μm, about 800 μm, about 900 μm, or about 1000 μm. In some embodiments, the spacer creates a fully enclosed chamber around the biological sample (e.g., tissue sample or a region of interest) and/or the array. The fully enclosed spacer can be any shape. In some embodiments, the fully enclosed (e.g., encompassed) spacer is one of a square or a rectangle. In some embodiments, the spacer conforms to the shape of the biological sample.
In some embodiments, the spacer partially encloses the biological sample (e.g., tissue or region of interest) or the array. In some embodiments, the spacer surrounds the biological sample on one, two, or three sides. In some embodiments, the spacer partially encloses the biological sample, enclosing approximately at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of the surrounding biological sample.
In some embodiments, no spacer is used in the system and/or methods disclosed herein. In some embodiments, a spacer functions as a gasket as disclosed herein.
In some embodiments of any of the fluid delivery methods described herein, the gasket (e.g., any of the gaskets described herein, a spacer), including a virtual gasket, can be applied to a region of interest in a biological sample. In some embodiments, two or more gaskets, including two or more virtual gaskets, can be applied to 2, 3, 4, or more regions of interest in the biological sample. In some embodiments, a fluid (e.g., a permeabilization solution) can be delivered to 2, 3, 4, or more regions of interest where a gasket, including a virtual gasket, substantially encompasses a region of interest in the biological sample.
In some embodiments, the fluid includes permeabilization reagents (e.g., any of the permeabilization reagents described herein). In some embodiments, the rate of permeabilization of the biological sample is modulated by delivering the permeabilization reagents (e.g., a fluid containing permeabilization reagents) at various temperatures. For example, the fluid (e.g., a permeabilization solution, a buffer) can be delivered from about 5° C. to about 80° C., from about 10° C. to about 75° C., from about 15° to about 70° C., from about 20° C. to about 65° C., from about 25° C. to about 60° C., from about 30° C. to about 55° C., from about to about 50°, and from about 40° C. to about 45° C. In some embodiments, the permeabilization solution can be about 5° C., about 6° C., about 7°, about 8° C., 9°, about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23°, 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., about 30° C., about 31° C., about 32° C., about 33° C., about 34° C., about 35° C., about 36° C., about 37° C., about 38° C., about 39° C., about 40° C., about 41° C., about 42° C., about 43° C., about 44° C., about 45° C., about 46° C., about 47° C., about 48° C., about 49° C., about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., about 55° C., about 56° C., about 57° C., about 58° C., about 59° C., about 60° C., about 61° C., about 62° C., about 63° C., about 64° C., about 65° C., about 66° C., about 67° C., about 68° C., about 69° C., about 70° C., about 71° C., about 72° C., about 73° C., about 74° C., about 75° C., about 76° C., about 77° C., about 78° C., about 79° C., or about 80° C.
In some embodiments, the permeabilization reagents are dried permeabilization reagents. In some embodiments, the dried permeabilization reagents are disposed on a substrate (e.g., the first substrate, the second substrate). In some embodiments, delivering the fluid (e.g., by any of the fluid delivery methods described herein) solubilizes the dried permeabilization reagents.
In some embodiments, controlling the temperate of the first substrate, the second substrate, or both, modulates permeabilization of the biological sample. For example, the first substrate, the second substrate, or both, can be disposed in a substrate holder (e.g., any of the substrate holders described herein). In some embodiments, heating the first substrate, the second substrate, or both includes heating the permeabilization solution (e.g., the fluid comprising permeabilization reagents, solubilized dried permeabilization reagents) and modulating permeabilization of the biological sample. For example, permeabilization can be actuated by heating once the system has equilibrated (e.g., after fluid delivery) and there is no flow present in the system. In some embodiments, cooling the first substrate, the second substrate, or both includes cooling the permeabilization solution (e.g., the fluid including permeabilization reagents, solubilized dried permeabilization reagents) and modulating permeabilization of the biological sample.
In some embodiments, the temperature of the first and second members is lowered to a first temperature that is below room temperature (e.g., 25 degrees Celsius) (e.g., 20 degrees Celsius or lower, 15 degrees Celsius or lower, 10 degrees Celsius or lower, 5 degrees Celsius or lower, 4 degrees Celsius or lower, 3 degrees Celsius or lower, 2 degrees Celsius or lower, 1 degree Celsius or lower, 0 degrees Celsius or lower, −1 degrees Celsius or lower, −5 degrees Celsius or lower). In some embodiments, the sample holder includes a temperature control system (e.g., heating and cooling conducting coils) that enables a user to control the temperature of the sample holder. Alternatively, in other embodiments, the temperature of the sample holder is controlled externally (e.g., via refrigeration or a hotplate). In a first step, the second member, set to or at the first temperature, contacts the first substrate, and the first member, set to or at the first temperature, contacts the second substrate, thereby lowering the temperature of the first substrate and the second substrate to a second temperature. In some embodiments, the second temperature is equivalent to the first temperature. In some embodiments, the first temperature is lower than room temperature (e.g., 25 degrees Celsius). In some embodiments, the second temperature ranges from about −10 degrees Celsius to about 4 degrees Celsius. In some embodiments, the second temperature is below room temperature (e.g., 25 degrees Celsius) (e.g., 20 degrees Celsius or lower, 15 degrees Celsius or lower, 10 degrees Celsius or lower, 5 degrees Celsius or lower, 4 degrees Celsius or lower, 3 degrees Celsius or lower, 2 degrees Celsius or lower, 1 degree Celsius or lower, 0 degrees Celsius or lower, −1 degrees Celsius or lower, −5 degrees Celsius or lower).
In some embodiments, controlling the temperate of the first substrate, the second substrate, or both modulates permeabilization of the biological sample includes heating to about 25° C. to about 55° C., to about 30° C. to about 50° C., to about 35° C. to about 45° C., or to about 40° C. In some embodiments, controlling the temperate of the first substrate, the second substrate, or both modulates permeabilization of the biological sample includes heating to about 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., or 55° C.
In some embodiments, the biological sample is permeabilized for a period of time. For example, the biological sample can be permeabilized from about 1 minute to about 90 minutes or any length of time in between, from about 5 minutes to 85 minutes, from about 10 minutes to about 80 minutes, from about 15 minutes to about 75 minutes, from about 20 minutes to about 70 minutes, from about 25 minutes to about 65 minutes, from about 30 minutes to about 60 minutes, from about 35 minutes to about 55 minutes, from about 40 minutes to about 50 minutes, or about 45 minutes. In some embodiments, the biological sample is permeabilized for about 30 minutes at 40° C.
In some embodiments, analytes that are released from the permeabilized tissue of the sample diffuse to the surface of the first substrate and are captured on the feature array (e.g., barcoded probes) of the second substrate. In a subsequent step, the first substrate and the second substrate are separated (e.g., pulled apart) and temperature control is stopped.
In some embodiments, the biological sample is imaged. In some embodiments, the biological sample is imaged on the first substrate prior to alignment with the second substrate. In some embodiments, the biological sample is a tissue section. In some embodiments, the biological sample is a fresh frozen biological sample. In some embodiments, the fresh frozen biological sample is a fresh frozen tissue section. In some embodiments, the biological sample is a fixed biological sample. In some embodiments, the fixed biological sample is a formalin-fixed paraffin-embedded biological sample.
In some embodiments, the array includes a plurality of features. In some embodiments, the array includes about 5,000 features. In some embodiments, a feature of the plurality of features is a bead. In some embodiments, a plurality of capture probes are attached to the bead. In some embodiments, the capture probe comprises a capture domain, and a spatial barcode unique to the feature. In some embodiments, the capture domain of the capture probe includes a poly(T) sequence. In some embodiments, the capture probe includes one or more functional domains, a cleavage domain, a unique molecular identifier, and combinations thereof.
Also provided herein are kits including a first substrate including a coating (e.g., any of the coatings described herein) for adhering a biological sample, a second substrate comprising an array, and a spacer. Also provided herein are kits including a first substrate, the first substrate comprising a surface for adhering a biological sample, a second substrate comprising an array, and a spacer. In some kits, the spacer is disposed on the first substrate and/or the second substrate. In some kits, the spacer at least partially surrounds the biological sample and/or the array. In some kits, the spacer may be disposed between the first substrate and second substrate and configured to maintain a fluid within a chamber comprising the first substrate, the second substrate, the biological sample, and the spacer. The spacer may be further configured to maintain a separation distance between the first substrate and the second substrate. In some kits, the kit includes a paraffin-wax crayon. In some kits, the kit includes a hydrophobic coating stamp. In some kits, the kit includes a reverse transcriptase and a nuclease.
In some embodiments, the example workflows and the example sample handling apparatuses described herein may allow for quick and efficient spatial analysis for multiple tissue/biological samples. The workflows and the example workflows and the example sample handling apparatuses described herein may facilitate multiple sandwich closings between a first and a second substrate. In some aspects, the sandwich closings may be performed in series or in parallel to achieve spatial analysis of multiple tissue/biological samples.
In some aspects, the example sample handling apparatuses described herein may implement software to provide some of the functions of the sample handling apparatus. For example, software may be used to control aspects of image processing, substrate alignment, substrate temperature control, instrument safety, or the like.
In some aspects, during the step 4308, the sample handling apparatus may capture an image 4307 of the tissue section. The image may include a low resolution image of the tissue section and any fiducial on the tissue slide. At step 4310, the workflow 4300 may include performing reverse transcription on the second substrate. Step 4312 may include performing second strand synthesis on the second substrate. At step 4314, the workflow 4300 may include generating a cDNA library associated with a particular spatial barcode of the gene expression slide. The sequence step 4316 may include library amplicons may be sequenced and analyzed to decode spatial information. Barcoded cDNA libraries may be mapped back to a specific spot on a capture area of the capture probes 306. This gene expression data may be subsequently layered over a high-resolution microscope image of the tissue section. In some aspects, during or before sequencing, a high resolution image 4317 of the tissue section may be captured for the layering described above.
The sample handling apparatus 400 also includes a processor 4620, a memory 4625, an input device 4630, and a display 4635. The processor 4620 can be configured to execute computer-readable instructions stored the memory 4625 to perform the workflows and processes described herein. The processor 4620 can also execute computer-readable instructions stored in the memory 4625, which cause the processor 4620 to control operations of the sample handling apparatus 400 via the I/O control board 4605 and/or the image capture device 2120 via the camera control 4610. In this way, the processor 4620 can control an operation of the sample handling apparatus 400 to align a sample with an array. For example, the processor 4620 can execute instructions to cause either of the first retaining mechanism or the second retaining mechanism to translate within the sample handling apparatus 400 so as to adjust their respective locations and to cause a sample area of a first substrate to be aligned with an array area of a second substrate.
The input device 4630 can include a mouse, a stylus, a touch-pad, a joy stick, or the like configured to receive user inputs from a user. For example, a user can use the input device 4630 to provide an input indicating a sample area indicator for a first substrate. The display 4635 can include a graphical user interface 4640.
The network interface 4615 may be configured to provide wired or wireless connectivity with (e.g., via Ethernet, Wi-Fi, or the like) a network 4645, such as the Internet, a local area network, a wide area network, a virtual private network, or the like. The network 4645 may be connected to one or more distributed computing resources, such as a cloud computing environment, a software as a service (SaaS) pipeline 4650, and/or a support portal 4655. The SaaS pipeline 4650 may be configured to aid or control automated image alignment or other alignment. The support portal 4655 may be configured to send images/videos/logs to the support portal and for issues to debug. The sample handling apparatus 400 can also be communicatively coupled via the network 4645 to a second computing device 4660 located remotely from the location of the sample handling apparatus 400.
The trough 4805 can prevent excess media supplied during permeabilization from entering gaps between a slide and the retaining mechanism 1422. The trough 4805 can form a gap that can suppress capillary flow pumping and reduce fluid movement during application of a reagent or a permeabilization solution and the entire assay time as described herein. The trough 4805 can advantageously suppress and manage the flow of fluid reagents used in the sample handling apparatus 1400.
The trough 4815 can prevent excess media supplied during permeabilization from entering gaps between a slide and the retaining mechanism 1408. The trough 4815 can form a gap that can suppress capillary flow pumping and reduce fluid movement during application of a reagent or a permeabilization solution and the entire assay time as described herein. The trough 4815 can advantageously suppress and manage the flow of fluid reagents used in the sample handling apparatus 1400.
In some embodiments, the retaining mechanism 1408 can include alignment marks 4905. The alignment marks 4905 can be provided for substrates with larger sample areas, such as an 11 mm×11 mm sample area. In some embodiments, the retaining mechanism can include alignment marks 4910. The alignment marks 4910 can be provided for a second substrate with a second sample area size, such as a 6.5 mm×6.5 mm sample area size. In some embodiments, the retaining mechanism can include alignment marks 4915. The alignment marks 4915 can indicate an exclusion zone in which a sample on a substrate should not be located. In some embodiments, the alignment marks 4915 can define an exclusion zone associated with raised or elevated substrate features, such as a label or one or more frosted portions of a substrate.
As further shown in
The leveling interfaces 5205, 5210, and 5215 can be provided in the display 4635 when the sample handling apparatus is in leveling mode.
As further shown in
Analyte capture onto spatially barcoded arrays and subsequent sequencing was demonstrated under sandwich and non-sandwich conditions. For the test (sandwiching) condition, archived tissue-mounted standard glass slides containing hematoxylin/eosin stained fresh frozen mouse brain sections were used. For control (non-sandwich) condition, array substrate slides (e.g., GEx array slides) with hematoxylin/eosin stained fresh frozen mouse brain sections mounted directly onto the array area were used. Under both conditions, tissue sections were subjected to a hematoxylin destaining step. Slides processed according to the “sandwiching” condition were briefly dried at 37° C., then mounted in an instrument along with a GEx slide and a permeabilization buffer comprising sarkosyl and proteinase K. Upon sandwich closure in the instrument, the tissue sections were permeabilized for 1 minute. For the tissue-mounted GEx slides processed according to the non-sandwich condition, sections were permeabilized for 5 minutes using the same permeabilization buffer without sandwiching. For both conditions, following permeabilization, captured polyA-containing mRNA transcripts on the GEx slides were reverse transcribed into cDNA, followed by standard sequencing library preparation and sequencing.
Results depicting median genes per spot and median UMI counts per spot are shown in
Visual heat map results showing Log 10 UMIs are shown in
Spatial clustering analysis (top row 5805) and analysis of hippocampal transcript Hpca (bottom row 5810) are depicted in
In some embodiments, instead of sandwiching the substrate including a sample of tissue with the substrate including the array of spatial barcodes described herein, the substrate including a sample of tissue can be sandwiched with a tissue optimization assay substrate which has a distributed area containing a plurality of PolyT capture probes. mRNA transcripts can be reverse transcribed in the presence of fluorescently labeled nucleotides, which can result in fluorescent cDNA linked to the capture probes. The linked cDNA can then be imaged. Image brightness and image sharpness can provide indications of the degree to which permeabilization and transcript capture was accomplished.
In one example aspect, an angled closure (e.g., see workflow 1700 and
This example provides an exemplary method for integrating immunostaining into a sandwich assembly workflow as described herein. In a non-limiting example, a biological sample is sectioned and placed on a first slide. After fixing (e.g., with 2% formalin or with methanol) and blocking (e.g., with Triton-X), the biological sample is subjected to an antibody incubation step. Following the antibody incubation step, the method further comprises an antibody staining step. The antibody staining step can include a direct method of immunostaining in which a labelled antibody binds directly to the analyte being stained for. Alternatively, the antibody staining step can include an indirect method of immunostaining in which a first antibody binds to the analyte being stained for, and a second, labelled antibody binds to the first antibody. Following the antibody staining step, the sample is imaged, e.g., by immunohistochemistry or immunofluorescence. Following imaging, a second slide comprising a feature array is placed in proximity to the first slide, creating a sandwich configuration. Permeabilization occurs while the slides are in the sandwich configuration.
Exemplary permeabilization conditions can include permeabilization with pepsin, or permeabilization with proteinase K and SDS. Analytes migrate to and are captured by probes on the second slide, then extended to capture the complementary sequence of the captured oligonucleotides and analytes. Following permeabilization, the sandwich is disassembled and the extended capture probe is then amplified and sequenced according to any one of the methods described herein. Subsequent sequence analysis is then used to determine spatial information regarding the analytes captured from the tissue sample.
In some aspects, alignment of the first substrate and the second substrate may be improved with an articulating first member 404.
In some aspects, alignment of the first substrate and the second substrate may be improved with a fixed second member 410.
In some aspects, the movement of the first members 7005A and/or 7005B may occur after a user manually closes the first members 7005A and/or 7005B over the second member 7010. As further shown, the image capture device 1420 may be positioned inferior to (e.g., below) the second member 7010. As further shown, the image capture device 1420 may be positioned inferior to (e.g., below) the second member 7010. This position may allow the image capture device 1420 to capture consistent in-focus images of the first substrate 303, the tissue sample 302, the second substrate 304, and/or the capture probes 306 at different time periods of a sandwich configuration analysis. In some aspects, a lighting source may positioned on, or superior to (e.g., above) the first members 7005A and/or 7005B to aid in image capture. Further, the fixed second member 7010 may allow the reagent droplet (e.g., permeabilization solution 305) to remain on the second substrate 304 more easily than if the second member articulated and/or changed angles. In some aspects, moving the first members 7005A and/or 7005B along the z axis as opposed to the second member 7010 may allow single cell analysis and a thinner spacer (e.g., spacers 3610, 6805). In some aspects, an array indicator (not shown) may be disposed on the first member 7005A and/or 7005B and/or the second member 7010. The array indicator may be installed using laser engraving.
As further shown in
In some embodiments, the sample handling apparatus described herein can include components and functionality configured to provide accurate control and positioning of the top portion of the sample handling apparatus (e.g., the top portion 7025 described in relation to
In some embodiments, the “home” position of the top portion 7410 of the sample handling apparatus 7400 can be additionally or alternatively determined using an impedance sensor coupled to a motor controlling the closure of the top portion 7410 with respect to the bottom portion 7415. The impedance sensor can measure the impedance of the motor at the start of closing the top portion 7410 toward the bottom portion 7415 and can assign a “zero” value, corresponding to the “home” position when the top portion 7410 contacts a physical bump or stop configured in the bottom portion 7415 In this way, the “zero” value can correspond to the “home” position and articulation of the top portion 7410 via the motor can be performed relative to the “zero” value.
The subject matter described herein may be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration. The implementations set forth in the foregoing description do not represent all implementations consistent with the subject matter described herein. Instead, they are merely some examples consistent with aspects related to the described subject matter. Although a few variations have been described in detail above, other modifications or additions are possible. In particular, further features and/or variations may be provided in addition to those set forth herein. For example, the implementations described above may be directed to various combinations and subcombinations of the disclosed features and/or combinations and subcombinations of several further features disclosed above. In addition, the logic flows depicted in the accompanying figures and/or described herein do not necessarily require the particular order shown, or sequential order, to achieve desirable results. Other implementations may be within the scope of the following claims.
One or more aspects or features of the subject matter described herein may be realized in digital electronic circuitry, integrated circuitry, specially designed ASICs, field programmable gate arrays (FPGAs) computer hardware, firmware, software, and/or combinations thereof. These various aspects or features may include implementation in one or more computer programs that are executable and/or interpretable on a programmable system including at least one programmable processor, which may be special or general purpose, coupled to receive data and instructions from, and to transmit data and instructions to, a storage system, at least one input device, and at least one output device. The programmable system or computing system may include clients and servers. A client and server are generally remote from each other and typically interact through a communication network. The relationship of client and server arises by virtue of computer programs running on the respective computers and having a client-server relationship to each other.
These computer programs, which may also be referred to as programs, software, software applications, applications, components, or code, include machine instructions for a programmable processor, and may be implemented in a high-level procedural and/or object-oriented programming language, and/or in assembly/machine language. As used herein, the term “machine-readable medium” refers to any computer program product, apparatus and/or device, such as for example magnetic discs, optical disks, memory, and Programmable Logic Devices (PLDs), used to provide machine instructions and/or data to a programmable processor, including a machine-readable medium that receives machine instructions as a machine-readable signal. The term “machine-readable signal” refers to any signal used to provide machine instructions and/or data to a programmable processor. The machine-readable medium may store such machine instructions non-transitorily, such as for example as would a non-transient solid-state memory or a magnetic hard drive or any equivalent storage medium. The machine-readable medium may alternatively or additionally store such machine instructions in a transient manner, such as for example, as would a processor cache or other random access memory associated with one or more physical processor cores.
To provide for interaction with a user, one or more aspects or features of the subject matter described herein may be implemented on a computer having a display device, such as for example a cathode ray tube (CRT) or a liquid crystal display (LCD) or a light emitting diode (LED) monitor for displaying information to the user and a keyboard and a pointing device, such as for example a mouse or a trackball, by which the user may provide input to the computer. Other kinds of devices may be used to provide for interaction with a user as well. For example, feedback provided to the user may be any form of sensory feedback, such as for example visual feedback, auditory feedback, or tactile feedback; and input from the user may be received in any form, including acoustic, speech, or tactile input. Other possible input devices include touch screens or other touch-sensitive devices such as single or multi-point resistive or capacitive track pads, voice recognition hardware and software, optical scanners, optical pointers, digital image capture devices and associated interpretation software, and the like.
In the descriptions above and in the claims, phrases such as “at least one of” or “one or more of” may occur followed by a conjunctive list of elements or features. The term “and/or” may also occur in a list of two or more elements or features. Unless otherwise implicitly or explicitly contradicted by the context in which it used, such a phrase is intended to mean any of the listed elements or features individually or any of the recited elements or features in combination with any of the other recited elements or features. For example, the phrases “at least one of A and B;” “one or more of A and B;” and “A and/or B” are each intended to mean “A alone, B alone, or A and B together.” A similar interpretation is also intended for lists including three or more items. For example, the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B together, A and C together, B and C together, or A and B and C together.” Use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US21/50931 | 9/17/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63153211 | Feb 2021 | US | |
| 63106779 | Oct 2020 | US | |
| 63080514 | Sep 2020 | US |
