Refractive index matching formulations
Refractive index matching (RIM) formulations, their use for mounting biological specimens (e.g., cells and tissues) to a substrate, and methods of visualizing biological specimens embedded in RIM formulations are described.
1. A refractive index matching (RIM) solution, comprising:
a) a water-soluble polymer, wherein the polymer has a molar refraction (RLL) to molar volume (Vm) ratio of 0.27 to 0.34, wherein the water-soluble polymer is uncharged, wherein the uncharged, water-soluble polymer comprises a monomer residue of N—R acrylamide or N—R methacrylamide, wherein R is methyl, ethyl, propyl, isopropyl or N,N-dimethylacrylamide, N,N-dimethylmethacrylamide, N,N-diethylacrylamide, N, N-dimethylmethacrylamide; N-2-hydroxypropyl methacrylamide, or a combination thereof,
b) a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.318:1 to 4:1, and
c) water or a buffer, wherein the RIM solution has a refractive index of 1.36 or greater.
2. The RIM solution of claim 1 , wherein the uncharged, water-soluble polymer is poly(N-methyl methacrylamide) (PMMAm).
3. The RIM solution of claim 1 , wherein the refractive index is 1.60 or less.
4. A method of mounting a biological specimen on a substrate, comprising:
depositing the biological specimen on the substrate; and
contacting the biological specimen with the RIM solution of claim 1 to provide a mounted sample.
5. The method of claim 4 , further comprising drying the mounted sample, whereby the water-soluble polymer solidifies to provide a solidified polymer.
6. The method of claim 5 , wherein the water-soluble polymer solidifies in about 48 hours or less at room temperature.
7. The method of claim 5 , wherein the refractive index of the solidified polymer exceeds 1.47.
8. The RIM solution of claim 1 , wherein the water-soluble polymer has a weight average molecular weight of about 1 kDa to about 100 kDa.
9. The RIM solution of claim 1 , further comprising an anti-oxidant.
10. The RIM solution of claim 1 , wherein the weight ratio of the water-soluble polymer to the polyol is 0.5:1 to 4:1.
11. The RIM solution of claim 1 , wherein the polyol is glycerol, diglycerol, polyglycerol, mannitol, sorbitol, thiodiethanol, thiodipropanol, or a combination thereof.
12. A kit for mounting a biological specimen on a substrate, comprising:
a) the refractive index matching (RIM) solution of claim 1 ; and
b) instructions for mounting the biological specimen on the substrate.
13. A refractive index matching (RIM) solution, comprising:
a) a water-soluble polymer, wherein the polymer has a molar refraction (RLL) to molar volume (Vm) ratio of 0.27 to 0.34, wherein the water-soluble polymer is charged;
b) a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.318:1 to 4:1; and
c) water or a buffer, wherein the RIM solution has a refractive index of 1.36 or greater.
14. The RIM solution of claim 13 , wherein the charged, water-soluble polymer is polyacrylic acid, polymethacrylic acid, poly(diallyldimethylammonium chloride), poly(sodium-4-styrenesulfonate), poly(ethyleneimine), poly(N,N-dimethylaminoethyl acrylate), poly(N,N-diethylethylamino acrylate), poly(allylamine), poly[bis(2-chloroethyl)ether-co-1,3-bis[3-(dimethylamino)propyl]urea], or poly(vinylsulfonic acid, sodium salt).
15. A solidified mountant, comprising a water-soluble polymer, wherein the polymer has a molar refraction (RLL) to molar volume (Vm) ratio of 0.27 to 0.34, and a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.318:1 to 4:1, and wherein the solidified mountant has a refractive index of 1.48 or greater, wherein the water-soluble polymer is (i) a charged water-soluble polymer or (ii) an uncharged, water-soluble polymer that comprises a monomer residue of N—R acrylamide or N—R methacrylamide, wherein R is methyl, ethyl, propyl, isopropyl or H; N,N-dimethylacrylamide, N,N-dimethylmethacrylamide, N,N-diethylacrylamide, N, N-diethylmethacrylamide, N-2-hydroxypropyl methacrylamide, or a combination thereof.
16. The solidified mountant of claim 15 , wherein the weight ratio of the water-soluble polymer to the polyol is 0.5:1 or greater.
17. The solidified mountant of claim 15 , wherein the refractive index is 1.48 to 1.54.
18. The RIM solidified mountant of claim 15 , wherein the refractive index of the solidified mountant matches the refractive index of soda-lime glass, borosilicate glass, or immersion oil.
19. The solidified mountant of claim 15 , wherein the weight ratio of the water-soluble polymer to the polyol is 0.5:1 to 4:1, and wherein the solidified mountant has a refractive index of 1.48 to 1.60.
20. A mounted biological specimen, comprising a biological specimen disposed on a substrate, wherein the biological specimen is embedded in the solidified mountant of claim 15 .