Compositions and methods for clearing a biological sample
The disclosure provides improved materials and methods for optically clearing biological tissue that is subsequently used for deep tissue imaging analysis. Also provided is a description of a microscopic image acquisition methodology in which imagery of intact tissues are acquired to rapidly acquire microscopy data on a whole-organ scale to maximize cost effectiveness for biological microscopy and minimize time spent performing such analysis.
1. A method for removing lipid from a biological sample, the method comprising:
contacting a fixed biological sample with a composition in an amount and for a time sufficient to remove lipid, the composition comprising:
sodium dodecyl sulfate (SDS), 3-(N,N-Dimethylmyristylammonio)propanesulfonate (SB3-14), polysorbate 20, t-octylphenoxypolyethoxyethanol, sodium deoxycholate, and a salt;
wherein the contacting results in the sample being significantly free of lipid.
2. The method of claim 1 , wherein the composition comprises about 1% to about 10% (weight:volume) of SDS.
3. The method of claim 1 wherein the composition comprises about 0.03% weight:volume (w/v) to about 3% w/v SB3-14.
4. The method of claim 1 wherein the composition comprises about 0.3% weight:volume (w/v) to about 3% w/v polysorbate 20.
5. The method of claim 1 wherein the composition comprises about 0.3% weight:volume (w/v) to about 3% w/v t-octylphenoxypolyethoxyethanol.
6. The method of claim 1 wherein the composition comprises about 0.1% weight:volume (w/v) to about 1% w/v sodium deoxycholate.
7. The method of claim 1 wherein the composition has a pH of from about 7 to about 9.
8. The method of claim 7 wherein the composition has a pH of from about 7.8 to about 8.8.
9. The method of claim 8 wherein the composition has a pH of from about 8.3 to about 8.5.
10. The method of claim 1 , wherein the salt is selected from the group consisting of sodium chloride, calcium chloride and sodium metaborate.
11. The method of claim 10 , wherein the salt is present in the composition at a concentration of from about 50 mM to about 500 mM.
12. The method of claim 11 , wherein the salt is present in the composition at a concentration of 150 mM.
13. The method of claim 1 , wherein the composition does not comprise boric acid.
14. The method of claim 1 , wherein the composition does not comprise urea.
15. The method of claim 1 , wherein the biological sample is fixed by embedding the biological sample in a fixative comprising acrylamide, paraformaldehyde, and optionally saponin.
16. The method of claim 1 , wherein contacting comprises perfusing the biological sample with the composition.
17. The method of claim 16 , wherein the biological sample is contacted with an antibody.
18. The method of claim 1 , further comprising the step of contacting the biological sample with an imaging solution comprising 2,2′-thiodiethanol (TDE).
19. The method of claim 18 wherein the imaging solution comprises from about 1% to about 90% TDE.
20. The method of claim 1 , wherein the biological sample is obtained from a plant or a eukaryote.
21. The method of claim 20 wherein the biological sample is obtained from a eukaryote.
22. The method of claim 21 wherein the biological sample is an organ, a tissue, or a cell taken from a multicellular organism.
23. The method of claim 21 , wherein the eukaryote is a mouse embryo or a zebrafish.
24. The method of claim 22 , wherein the organ is selected from the group consisting of heart, blood vessels, salivary gland, esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, anus, endocrine gland, adrenal gland, kidney, ureter, bladder, lymph node, tonsils, adenoid, thymus, spleen, skin, muscle, brain, spinal cord, nerve, ovary, fallopian tube, uterus, vagina, mammary gland, testes, prostate, penis, pharynx, larynx, trachea, bronchi, lung, diaphragm, cartilage, ligaments, and tendon.
25. The method of claim 1 , further comprising imaging the biological sample.
26. The method of claim 25 , wherein the imaging is macroscale imaging.
27. The method of claim 25 , wherein the imaging provides three-dimensional information.
28. The method of claim 26 , wherein the macroscale imaging results in the ability to visualize the sample to a depth of about 5 μm, about 100 μm, about 200 μm, about 500 μm, about 1 mm, or about 10 mm.