Swellable and structurally homogenous hydrogels and methods of use thereof

1. A hydrogel that is the product of a non-radical polymerization reaction between a monomer of Formula (A1):

and a monomer of Formula (B1):

wherein:

each n is an integer greater than or equal to 1;

each p is an integer greater than or equal to 1;

Z + is a counter cation;

X and Y 1 are each crosslinkable moieties, and

X and Y 1 covalently crosslink to end-link the monomers.

2. The hydrogel of claim 1 , wherein X is a moiety comprising a terminal azide group and Y 1 is a moiety comprising a terminal alkyne, and wherein X and Y 1 crosslink by copper-free azide-alkyne cycloaddition.

3. The hydrogel of claim 1 , wherein X and Y 1 crosslink by amine-NHS ester reaction.

4. The hydrogel of claim 1 , wherein X and Y 1 crosslink by maleimide-thiol reaction.

5. The hydrogel of claim 1 , wherein X and Y 1 crosslink by trans-cyclooctene (TCO)-tetrazine reaction.

6. The hydrogel of claim 1 , wherein the hydrogel is labelled.

7. A composite comprising a biological sample and the hydrogel of claim 1 .

8. A method of preparing the composite of claim 7 , comprising permeating the biological sample with a monomer of Formula (A1) or a monomer of Formula (A3), and a monomer of Formula (B1) to form a hydrogel by non-radical polymerization.

9. A method of microscopy comprising:

a. permeating the biological sample with a monomer of Formula (A1):

and a monomer of Formula (B1):

to form a hydrogel according to claim 1 by non-radical polymerization;

b. isotropically expanding the composite by contacting it with an aqueous solution; and

c. viewing the expanded composite using microscopy;

wherein:

each n is an integer greater than or equal to 1;

each p is an integer greater than or equal to 1;

Z + is a counter cation

X and Y 1 are each crosslinkable moieties, and

X and Y 1 covalently crosslink to end-link the monomers.

10. A method for in-situ sequencing of target nucleic acids present in a biological sample comprising the steps of:

a. attaching target nucleic acids present in the sample with a molecule linker or nucleic acid adapter;

b. permeating the sample with a monomer of Formula (A1):

and a monomer of Formula (B1):

to form a hydrogel according to claim 1 by non-radical polymerization and thereby forming a sample-hydrogel complex, wherein the small molecule linker or nucleic acid adaptor is attached both to the target nucleic acids present in the sample and to the hydrogel;

c. digesting proteins present in the sample;

d. expanding the complex to form a first enlarged sample;

e. re-embedding the first enlarged sample in a non-swellable material to form a re-embedded complex;

f. modifying the target nucleic acids or the nucleic acid adaptor to form a target nucleic acids or a nucleic acid adaptor; and

g. sequencing the nucleic acids present in the re-embedded complex;

wherein:

each n is an integer greater than or equal to 1;

each p is an integer greater than or equal to 1;

Z + is a counter cation;

X and Y 1 are each crosslinkable moieties, and

X and Y 1 covalently crosslink to end-link the monomers.

11. A method for enlarging a biological sample for microscopy, the method comprising the steps of:

a) permeating a sample with a first hydrogel, wherein the sample is anchored to the swellable material;

b) swelling the swellable material resulting in a first expanded sample;

c) optionally permeating the first expanded sample with a second hydrogel; and

d) optionally swelling the second hydrogel resulting in a second expanded sample;

wherein the first hydrogel and/or the second hydrogel is the hydrogel of claim 1 .