Fluid delivery methods
Provided herein is a fluid delivery method for permeabilizing a biological sample. The method includes delivering the fluid to a first substrate and/or a second substrate. At least one of the first substrate and the second substrate includes a spacer. The method further includes assembling, subsequent to the delivering, a chamber comprising the first substrate, the second substrate, the biological sample, and the spacer. The spacer may be disposed between the first substrate and second substrate. The spacer 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 is disposed and/or at least partially surround the array disposed on the second substrate.
1 . A method for delivering a fluid to a biological sample, the method comprising:
delivering the fluid into an aperture between an inner wall and an outer wall of a spacer disposed on a first substrate and/or a second substrate, the first substrate comprising an area on which the biological sample is disposed, the second substrate comprising an array disposed thereon, and the spacer being positioned to at least partially surround the area of the first substrate and/or the array on the second substrate; and
assembling, subsequent to delivering fluid into the aperture, a chamber comprising the first substrate, the second substrate, the biological sample, and the spacer, such that:
the area of the first substrate, the spacer, and the second substrate at least partially enclose a volume comprising the biological sample, the assembled chamber is separate from the aperture and is at least partially surrounded by the inner wall of the spacer, and the spacer is positioned between the first substrate and second substrate and maintains a separation distance between the first substrate and the second substrate; and
at least a portion of the fluid delivered to the aperture is delivered to the chamber, and the spacer maintains within the chamber at least the portion of the fluid delivered to the aperture.
2 . The method of claim 1 , wherein the assembled chamber comprises a partially or fully sealed chamber.
3 . The method of claim 1 , wherein the separation distance comprises a distance of at least 2 μm.
4 . The method of claim 1 , wherein the separation distance comprises a distance between 5 μm to 25 μm.
5 . The method of claim 1 , wherein the second substrate comprises the spacer.
6 . The method of claim 1 , wherein the first substrate comprises the spacer.
7 . The method of claim 1 , wherein the fluid comprises a wetting agent.
8 . The method of claim 1 , wherein the fluid comprises one or more permeabilization reagent(s).
9 . The method of claim 1 , wherein:
assembling the chamber comprises delivering the fluid to a region of the first substrate and/or the second substrate at least partially defined by three sides of the spacer.
10 . The method of claim 1 , wherein:
delivering the fluid into the aperture comprises delivering the fluid to a region of the first substrate and/or the second substrate outside an enclosed area of the second substrate, the enclosed area formed by the spacer.
11 . The method of claim 1 , wherein:
delivering the fluid into the aperture comprises delivering the fluid to a region of the spacer outside an enclosed area of the first substrate and/or the second substrate, the enclosed area formed by the spacer.
12 . The method of claim 1 , wherein:
assembling the chamber comprises positioning the first substrate at an angle relative to the second substrate such that:
a dropped side of the first substrate contacts at least part of the fluid when the first substrate and the second substrate are within a threshold distance along an axis orthogonal to the second substrate, and
the dropped side of the first substrate urges at least the portion of the fluid from the aperture toward a side of the first substrate that is opposite the dropped side and into the chamber.
13 . The method of claim 1 , wherein:
assembling the chamber comprises assembling the chamber such that a hydrophobic pattern of the chamber at least partially surrounds the fluid and is positioned to at least partially surround the area on the first substrate and/or the array on the second substrate.
14 . The method of claim 1 , wherein the spacer is formed from a uniform thickness material.
15 . The method of claim 1 , wherein the spacer is formed from a material with a variable thickness.
16 . The method of claim 1 , wherein the spacer is printed on the first substrate and/or the second substrate.
17 . The method of claim 1 , wherein the spacer comprises a photoresist pattern.
18 . The method of claim 1 , wherein the spacer comprises a beveled edge on one or more sides of the spacer.
19 . The method of claim 1 , wherein the array on the second substrate comprises a plurality of discrete features each comprising a plurality of capture probes.
20 . The method of claim 19 , wherein a capture probe of the plurality of capture probes comprises (i) a capture domain and (ii) a spatial barcode unique to a feature of the plurality of discrete features.
21 . The method of claim 1 , wherein the biological sample is a tissue sample.
22 . The method of claim 1 , wherein the spacer comprises a material selected to promote a contact angle of at least 50 degrees.
23 . The method of claim 1 , wherein the spacer comprises a material selected from graphite, polyester, Polytetrafluoroethylene (PTFE), polyamide, polychlorotrifluoroethylene (PCTFE), poly(ethene-co-tetrafluoroethene) (ETFE), and PEEK (Polyether ether ketone).
24 . The method of claim 1 , wherein the spacer surrounds the area of the first substrate and/or the array on the second substrate.