Expansion microscopy
The present invention relates to an enlarged sample of interest for microscopy and methods for enlarging a sample of interest and the optical imaging of a sample of interest with resolution better than the classical microscopy diffraction limit, by synthesizing a swellable polymer network within a sample, it can be physically expanded, resulting in physical magnification.
1. A method for enlarging a cell or solid tissue-sample, the method comprising:
(a) labeling more than one biomolecule components of the sample with a trifunctional label, wherein the tri-functional label comprises an anchor capable of participating in polymerization and a visual label, further wherein the anchor is methacryloyl;
(b) permeating the sample with a composition comprising sodium acrylate, acrylamide and N,N-methylenebisacrylamide;
(c) polymerizing the composition within the sample to form a swellable material, wherein said polymerizing results in covalent crosslinking of the anchor to the swellable material to form a sample-swellable material complex; and
(d) adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex that results in an enlarged cell or tissue sample.
2. The method of claim 1 , wherein the sample-swellable material complex is swelled isotropically when the aqueous solvent or liquid is added, thereby maintaining the relative spatial relationship of the labeled biomolecules.
3. The method of claim 1 , wherein prior to performing the permeating step, the cell or tissue sample is treated with a detergent.
4. The method of claim 1 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
5. The method of claim 1 , wherein the aqueous solvent or liquid is water.
6. A microscopy method for producing a high-resolution image of a cell or a tissue sample, the method comprising:
enlarging the sample by performing the method of claim 1 ; and
(ii) viewing the enlarged sample under a microscope.
7. The method of claim 6 , wherein the sample-swellable material complex is swelled isotropically when the aqueous solvent or liquid is added, thereby maintaining the relative spatial relationship of the labeled biomolecules.
8. The method of claim 6 , wherein prior to the permeating step, the cell or tissue sample is treated with a detergent.
9. The method of claim 6 , wherein prior to the step of adding the aqueous solvent or liquid, the cell or tissue sample is subjected to digestion.
10. A method for optically imaging a cell or a tissue sample by physically enlarging the sample, the method comprising:
(i) performing the method of claim 1 , thereby achieving an enlarged cell or tissue sample; and
(ii) optically imaging the enlarged cell or tissue sample by viewing the sample under a microscope.
11. The method of claim 10 , wherein the sample-swellable material complex is swelled isotropically when the aqueous solvent or liquid is added, thereby maintaining the relative spatial relationship of the labeled biomolecules.
12. The method of claim 10 , wherein prior to the step of permeating, the cell or tissue sample is treated with a detergent.
13. The method of claim 10 , wherein prior to the step of adding the aqueous solvent or liquid, the cell or tissue sample is subjected to digestion.
14. A method for preparing an enlargeable cell or solid tissue sample, the method comprising:
(a) labeling more than one biomolecule components of the sample with a trifunctional label, wherein the tri-functional label comprises an anchor capable of participating in polymerization and a visual label, further wherein the anchor is methacryloyl;
(b) permeating the sample with a composition comprising sodium acrylate, acrylamide and N,N-methylenebisacrylamide;
(c) polymerizing the composition within the sample to form a polyelectrolyte hydrogel, wherein said polymerizing results in covalent crosslinking of the anchor to the polyelectrolyte hydrogel to form a cell or a tissue sample-polyelectrolyte hydrogel complex, thereby resulting in a sample prepared to be enlarged.
15. The method of claim 14 , wherein prior to the permeating step, the cell or tissue sample is treated with a detergent.
16. The method of claim 14 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
17. A method for isotropically enlarging a cell or solid tissue sample, the method comprising:
(a) labeling more than one biomolecule components of the sample with a label, wherein the label comprises a biomolecule binding moiety and a polymerizable ethylenically unsaturated moiety;
(b) permeating the sample with a composition comprising sodium acrylate, acrylamide and N,N-methylenebisacrylamide;
(c) polymerizing the composition within the sample to form a swellable material, wherein said polymerizing results in covalent crosslinking of the polymerizable moiety to the swellable material to form a sample-swellable material complex; and
(d) adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex that results in an isotropically enlarged tissue sample.
18. The method of claim 17 , wherein prior to performing the permeating step, the cell or tissue sample is treated with a detergent.
19. The method of claim 17 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
20. The method of claim 17 , wherein the aqueous solvent or liquid is water.
21. The method of claim 17 , further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope.
22. The method of claim 17 , further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope.
23. A method for isotropically enlarging a cell or solid tissue sample, the method comprising:
(a) labeling more than one biomolecule components of the sample with a trifunctional label, wherein the tri-functional label comprises an anchor capable of participating in polymerization and a visual label, further wherein the anchor is methacryloyl;
(b) permeating the sample with a composition comprising precursors of a crosslinked hydrogel comprising at least one polyelectrolyte monomer and a covalent crosslinker;
(c) polymerizing the composition within the sample to form a swellable material, wherein said polymerizing results in covalent crosslinking of the anchor to the swellable material to form a sample-swellable material complex; and
(d) adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex that results in an isotropically enlarged tissue sample.
24. The method of claim 23 , wherein prior to performing the permeating step, the cell or tissue sample is treated with a detergent.
25. The method of claim 23 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
26. The method of claim 23 , wherein the aqueous solvent or liquid is water.
27. The method of claim 23 , further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope.
28. The method of claim 23 , further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope.
29. A method for isotropically enlarging a cell or solid tissue sample, the method comprising:
(a) contacting the sample with a tag comprising a binding moiety and a polymerizable moiety, wherein the tag binds a biomolecule component of the sample;
(b) permeating the sample with a composition comprising precursors of a crosslinked hydrogel comprising at least one polyelectrolyte monomer and a covalent crosslinker;
(c) polymerizing the composition within the sample to form a swellable material, wherein said polymerizing results in covalent crosslinking of the tag to the swellable material to form a sample-swellable material complex; and
(d) adding an aqueous solvent or liquid to cause the sample-swellable material complex to swell, thereby physically expanding the complex that results in an isotropically enlarged tissue sample.
30. The method of claim 29 , wherein prior to performing the permeating step, the cell or tissue sample is treated with a detergent.
31. The method of claim 29 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
32. The method of claim 29 , wherein the aqueous solvent or liquid is water.
33. The method of claim 29 , further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope.
34. The method of claim 29 , further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope.
35. A method for isotropically enlarging a cell or solid tissue sample, the method comprising:
(a) contacting the sample with a tag comprising a binding moiety and a polymerizable moiety, wherein the tag binds a biomolecule component of the sample;
(b) permeating the sample with a composition comprising precursors of a polyelectrolyte hydrogel comprising at least one polyelectrolyte monomer and a covalent crosslinker;
(c) polymerizing the composition within the sample to form a polyelectrolyte hydrogel, wherein said polymerizing results in covalent crosslinking of the tag to the swellable material to form a sample- polyelectrolyte hydrogel complex; and
(d) adding an aqueous solvent or liquid to cause the sample-polyelectrolyte hydrogel complex to swell, thereby physically expanding the complex that results in an isotropically enlarged tissue sample.
36. The method of claim 35 , wherein prior to performing the permeating step, the cell or tissue sample is treated with a detergent.
37. The method of claim 35 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
38. The method of claim 35 , wherein the aqueous solvent or liquid is water.
39. The method of claim 35 , further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope.
40. The method of claim 35 , further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope.
41. A method for isotropically enlarging a cell or solid tissue sample, the method comprising:
(a) contacting the sample with a tag comprising a binding moiety and an anchor, wherein the binding moiety binds to biomolecule components of the sample; wherein the anchor comprises a polymerizable moiety;
(b) permeating the sample with a composition comprising at least one polyelectrolyte monomer and a covalent crosslinker;
(c) initiating polymerization to form a hydrogel comprising the at least one polyelectrolyte monomer, covalent crosslinker and anchor, wherein the hydrogel is covalently bound to the biomolecule components of the sample to form a sample- hydrogel complex; and
(d) adding an aqueous solvent or liquid to cause the sample-hydrogel complex to swell, thereby physically expanding the complex that results in an isotropically enlarged tissue sample.
42. The method of claim 41 , wherein prior to performing the permeating step, the cell or tissue sample is treated with a detergent.
43. The method of claim 41 , wherein prior to the step of adding the aqueous solvent or liquid to swell the sample-swellable material complex, the cell or tissue sample is subjected to digestion.
44. The method of claim 41 , wherein the aqueous solvent or liquid is water.
45. The method of claim 41 , further comprising the step of producing a high-resolution image of the sample by viewing the enlarged sample under a microscope.
46. The method of claim 41 , further comprising the step of optically imaging the enlarged sample by viewing the sample under a microscope.