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 tissue section, the method comprising:
delivering the fluid to a second substrate comprising a spatial array disposed thereon, the spatial array comprising a plurality of discrete features each comprising a plurality of capture probes attached to the second substrate, wherein a capture probe of the plurality of capture probes comprises (i) a capture domain and (ii) a spatial barcode unique to the feature, wherein the second substrate comprises a spacer disposed thereon, wherein the spacer is positioned to surround the spatial array; and
assembling, subsequent to the delivering, a first substrate comprising the tissue section and the second substrate, to form a chamber, wherein the spacer is disposed between the first substrate and the second substrate and is configured to maintain the fluid within the chamber and maintain a separation distance of less than 25 μm between the first substrate and the second substrate, the spacer further comprising a reservoir positioned within a wall of the spacer surrounding the chamber, the reservoir having an opening, wherein the fluid is delivered into the opening of the reservoir,
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 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 of the first substrate urging the fluid from the reservoir toward a side of the first substrate that is opposite the dropped side and into the chamber,
wherein the spacer comprises a material selected to promote a contact angle of at least 50 degrees,
and wherein the first substrate, the spacer, and the second substrate at least partially enclose a volume comprising the tissue section.
2. The method of claim 1 , wherein the first substrate, the spacer, and the second substrate fully enclose the volume comprising the tissue section.
3. The method of claim 1 , wherein the chamber comprises a partially or fully sealed chamber.
4. The method of claim 1 , wherein the separation distance comprises a distance of at least 2 μm.
5. The method of claim 1 , wherein the separation distance comprises a distance between about 5 μm to 25 μm.
6. The method of claim 1 , wherein the fluid comprises one or more permeabilization reagent(s).
7. The method of claim 1 , wherein the opening of the reservoir is positioned on a side of the spacer opposite of a side of the first substrate comprising the tissue section when the chamber is formed.
8. The method of claim 1 , wherein the spacer is formed from a uniform thickness material.
9. The method of claim 1 , wherein the spacer is printed on the second substrate or is attached to the second substrate via an adhesive.
10. The method of claim 1 , wherein the spacer comprises a photoresist.
11. The method of claim 1 , wherein the spacer comprises a beveled edge on one or more sides of the spacer.
12. The method of claim 1 , wherein the capture probe further comprises a functional domain, a cleavage domain, a unique molecular identifier, or any combination thereof.
13. The method of claim 1 , further comprising:
migrating one or more molecules from the tissue section toward the plurality of capture probes via the fluid; and
binding the one or more molecules from the tissue section to one or more capture probes of the plurality of capture probes.
14. The method of claim 13 , wherein the migrating comprises active migration via electrophoresis.
15. The method of claim 13 , wherein the migrating comprises passive migration via diffusion.
16. The method of claim 13 , wherein the binding occurs via hybridization.
17. The method of claim 13 , wherein the one or more molecules comprise nucleic acids.
18. The method of claim 1 , wherein a feature of the plurality of features is a spot.
19. The method of claim 1 , wherein the capture domain of the capture probe comprises a poly(T) sequence.
20. 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).