Expansion microscopy methods and kits
Provided herein are improved expansion microscopy methods. Also provided herein are kits useful in expansion microscopy.
1. A method of preparing a cell or tissue sample for microscopy, comprising:
permeating the cell or tissue sample with a gelling solution comprising an acrylate monomer, a methacrylate monomer, an acrylamide monomer, and/or a methacrylamide monomer for producing a water-swellable (co)polymer and an enal, wherein the enal is able to polymerize with the acrylate monomer, the methacrylate monomer, the acrylamide monomer, and/or the methacrylamide monomer; and
polymerizing the acrylate monomer, the methacrylate monomer, the acrylamide monomer, and/or the methacrylamide monomer of gelling solution with the enal of the gelling solution to form a swellable material containing the cell or tissue sample, wherein the enal of the swellable material covalently links the swellable material to a biomaterial in the sample,
wherein the covalent link between the enal of the swellable material and the biomolecule in the cell or tissue sample is reversible.
2. The method of claim 1 , wherein the gelling solution further comprises a crosslinker,
wherein the crosslinker comprises N,N′-methylene-bis(acrylamide).
3. The method of claim 2 , comprising from 0.01 g/100 mL to 5 g/100 mL of the crosslinker.
4. The method of claim 1 , further comprising homogenizing the cell or tissue sample after formation of the swellable material to facilitate even expansion of the sample; and hydrating the cell or tissue sample to cause the swellable material to physically expand.
5. The method of claim 4 , wherein the cell or tissue sample are homogenized by digesting the sample with a protease or by incubating the sample with a detergent and/or a denaturant,
wherein the detergent and/or the denaturant comprises 10 mM-300 mM SDS, 1M-8M urea, 1-100 mM mercaptoethanol/DTT, and/or 0.5-100 mM EDTA/EGTA.
6. The method of claim 4 , further comprising, prior to permeating the cell or tissue sample with the gelling solution, after polymerizing the acrylate monomer, the methacrylate monomer, the acrylamide monomer, and/or the methacrylamide monomer of gelling solution with the enal of the gelling solution, or after hydrating the cell or tissue sample, labeling a biomolecule of the cell or tissue sample with a detectable ligand, wherein the detectable ligand comprises an immunofluorescence label, an immunohistochemistry label, or a dye-conjugated or enzyme-conjugated ligand that optionally comprises a fluorescent dye.
7. The method of claim 6 , wherein the detectable ligand is an antibody, an antibody fragment, an antibody mimetic, a nucleic acid, or a nucleic acid analog.
8. The method of claim 4 , wherein during hydration, the cell or tissue sample swells isotropically, thereby maintaining the relative spatial relationship of biomolecules in the cell or tissue sample.
9. The method of claim 4 , wherein the cell or tissue sample comprises a cell wall, and the method further comprises, after formation of the swellable material and before hydrating the cell or tissue sample, enzymatically-digesting the cell wall with a glucanase or a cellulase,
wherein the glucanase or the cellulase comprises lyticase, chitinase, or lysozyme.
10. The method of claim 1 , wherein the monomers of the gelling solution are acrylic acid, an alkaline metal salt of an acrylate, methacrylic acid, an alkaline metal salt of a methyacrylate, acrylamide, and/or methacrylamide.
11. The method of claim 1 , wherein the enal is acrolein or methacrolein.
12. The method of claim 1 , wherein the sample is a section prepared from a whole organism or embryo.
13. The method of claim 1 , wherein the sample is a section prepared from a plurality of organs of an organism or a tissue section.
14. The method of claim 13 , wherein the sample comprises a tissue section and wherein the tissue section is provided mounted with a coverslip and/or paraffin-embedded, and the method further comprises pre-processing the tissue section with an organic solvent to remove the coverslip and/or paraffin from the sample, and replacing the organic solvent with an aqueous solution.
15. The method of claim 14 , wherein the organic solvent comprises xylene.
16. The method of claim 1 , further comprising staining the cell or tissue sample, wherein the stain is a DAPI stain.
17. The method of claim 1 , wherein the cell or tissue sample is fixed in a fixative, and the method further comprising removing the fixative from the cell or tissue sample by exposing the cell or tissue sample to a solvent for the fixative prior to permeating the cell or tissue sample with the gelling solution.
18. The method of claim 1 , comprising:
permeating the cell or tissue sample with a gelling solution comprising sodium acrylate, acrylamide, N,N′-methylene bis(acrylamide) and either acrolein or methacrolein;
polymerizing the sodium acrylate, acrylamide, and N,N′-methylene bis(acrylamide) of the gelling solution with the acrolein or the methacrolein of the gelling solution to form the swellable material containing the cell or tissue sample, wherein the acrolein or the methacrolein of the swellable material covalently links the swellable material to a biomaterial in the cell or tissue sample; and
hydrating the cell or tissue sample to cause the swellable material to physically expand.
19. The method of claim 1 , wherein the gelling solution comprises from 0.5M to 6M of acrylate monomer, methacrylate monomer, acrylamide monomer, and/or methacrylamide monomer and from 0.01 g/100 mL to 10 g/100 mL of the enal.
20. The method of claim 1 , wherein the enal comprises the following structure:
wherein R 1 and R 2 are independently H, C 1 -C 3 alkyl, C 1 -C 3 haloalkyl, C 1 -C 3 alkyl-aryl, or C 1 -C 3 halo alkyl-aryl.
21. A method of producing a high-resolution microscopic image of a cell or tissue sample comprising preparing a cell or tissue sample according to the method of claim 1 , and viewing and/or imaging the cell or tissue sample.