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 water-soluble polymer is (i) a charged water-soluble polymer; (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; or (iii) poly(methyl vinyl ether), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly(vinyl pyrrolidone) (PVP), or polyvinyl alcohol (PVA);
b) a polyol, wherein the polyol is polyvinyl alcohol, glycerol, diglycerol, polyglycerol, mannitol, sorbitol, erythritol, thiodiethanol, thiodipropanol, or a combination thereof, and wherein the weight ratio of the water-soluble polymer to the polyol is 0.125:1 to 4:1, wherein the water-soluble polymer and polyol are different; and
c) water or a buffer, wherein the RIM solution has a refractive index of about 1.36 to about 1.60.
2 . The RIM solution of claim 1 , wherein the water-soluble polymer has a molar refraction (R LL ) to molar volume (V m ) ratio of about 0.27 to about 0.34.
3 . The RIM solution of claim 1 , wherein the uncharged, water-soluble polymer is poly(N-methyl methacrylamide) (PMMAm), poly(acrylamide), or poly(methacrylamide).
4 . The RIM solution of claim 1 , 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).
5 . The RIM solution of claim 1 , further comprising an anti-oxidant.
6 . The RIM solution of claim 5 , wherein the anti-oxidant is 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, 3-carboxy proxyl, hydroquinone, mequinol, sodium sulfite, sodium erythorbate, ascorbic acid, propyl gallate, caffeic acid, paraphenylenediamine, 1,4-diazabicyclo[2.2.2]octane, or a combination thereof.
7 . A method of mounting a biological specimen on a substrate, comprising:
depositing the biological specimen on the substrate;
contacting the biological specimen with a refractive index matching (RIM) solution to provide a mounted sample, wherein the RIM solution comprises:
a) a water-soluble polymer, wherein the water-soluble polymer is (i) a charged water-soluble polymer; (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; or (iii) poly(methyl vinyl ether), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly(vinyl pyrrolidone) (PVP), or polyvinyl alcohol (PVA),
b) a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.125:1 to 4:1, wherein the water-soluble polymer and polyol are different, and
c) water or a buffer, wherein the RIM solution has a refractive index of about 1.36 to about 1.60; and
drying the mounted sample at room temperature to about 40° C. for about 48 hours or less to provide a solidified mountant.
8 . The method of claim 7 , wherein the RIM solution solidifies in about 24 hours or less at room temperature.
9 . The method of claim 7 , wherein the RIM solution solidifies in about 4 hours or less at a temperature of about 40° C.
10 . A solidified mountant, comprising a water-soluble polymer, and a polyol, wherein the weight ratio of the water-soluble polymer to the polyol is 0.125:1 to 4:1, wherein the water-soluble polymer is (i) a charged water-soluble polymer; (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; or (iii) poly(methyl vinyl ether), poly(2-ethyl-2-oxazoline), poly(2-methyl-2-oxazoline), poly(vinyl pyrrolidone) (PVP), or polyvinyl alcohol (PVA), wherein the solidified mountant has a refractive index of about 1.48 to about 1.60 and wherein the water-soluble polymer and polyol are different.
11 . The solidified mountant of claim 10 , wherein the polymer has a molar refraction (R LL ) to molar volume (V m ) ratio of about 0.27 to about 0.34.
12 . The solidified mountant of claim 10 , wherein the refractive index of the solidified mountant matches the refractive index of soda-lime glass, borosilicate glass, or immersion oil.
13 . The solidified mountant of claim 10 , wherein the uncharged, water-soluble polymer is poly(N-methyl methacrylamide) (PMMAm), poly(acrylamide), or poly(methacrylamide).
14 . The solidified mountant of claim 10 , 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 . The solidified mountant of claim 10 , wherein the polyol is polyvinyl alcohol, glycerol, diglycerol, polyglycerol, mannitol, sorbitol, erythritol, thiodiethanol, thiodipropanol, or a combination thereof.
16 . The solidified mountant of claim 10 , wherein the RIM solution further comprises an anti-oxidant.
17 . A mounted biological specimen, comprising a biological specimen disposed on a substrate, wherein the biological specimen is embedded in the solidified mountant of claim 10 .