METHOD FOR ADAPTING OPTICAL PROPERTIES OF AN IMMERSION MIXTURE, AND IMMERSION MIXTURES AND COMPONENTS THEREOF

Abstract

A method adapts optical properties of an immersion mixture by providing an immersion mixture made of multiple components, wherein at least two of the components have different optical properties and the immersion mixture has optical properties resulting from the proportions of the components, determining current actual values of the optical properties of the immersion mixture, and comparing the current actual values with target values. If actual values differ from target values by an unacceptable amount, a target mixture ratio of the components is determined to achieve the target values and the proportion of the components is set according to the target mixture ratio. For the components, water can be selected with a water-soluble inorganic compound or a water-soluble organic compound, or a non-water-soluble compound can be selected with a solvent suitable for dissolving the compound. Suitable chemical compounds and an immersion mixture made of multiple components can also be utilized.

Claims

1: A method for adapting optical properties of an immersion mixture, the method comprising: providing an immersion mixture comprising a plurality of components, wherein at least two of the plurality of components have optical properties different from one another and the immersion mixture possesses optical properties resulting from the respective proportions of the components, determining current actual values for the optical properties of the immersion mixture, comparing the current actual values with target values for optical properties of the immersion mixture, determining a target mixing ratio for the components of the immersion mixture where there is an impermissible difference between the actual values and the target values, wherein the target mixing ratio achieves the target values, and adjusting the respective proportions of the components in the immersion mixture in accordance with the target mixing ratio, wherein water is chosen as one of the components and a water-soluble inorganic compound or a water-soluble organic compound is chosen as a further component, or wherein a water-insoluble compound is chosen as one of the components and a solvent suitable for dissolution of the compound is chosen as a further component.

2: The method as claimed in claim 1, wherein at least two components having optical refractive power different from one another are used.

3: The method as claimed in claim 1, wherein the target values for the optical properties of the immersion mixture can be defined as a function of at least one determined optical property of a sample undergoing imaging.

4: An esterified perfluoropolyether of the chemical structure
R1-O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CF.sub.2O(CF.sub.2CF.sub.2O).sub.p(CF.sub.2O).sub.qCF.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.nO—R2 wherein the groups R1 and R2 are each either phenylacetate ester, tetrahydrofurancarboxylate ester, or (2-methoxyethoxy)acetate ester; n=1 to 3; and p and q are each chosen from within a range from 3 to 12.

5: A compound of the chemical structure ##STR00003## wherein the group R3 is a phenylacetate ester (radical).

6: An immersion mixture, comprising: a plurality of components, wherein at least two of the components have optical refractive powers different from one another, and the immersion mixture possesses an optical refractive power resulting from the respective proportions of the components, wherein one of the components is water and a further component is a water-soluble inorganic compound or a water-soluble organic compound, or wherein one of the components is a water-insoluble compound and a further component is a solvent suitable for dissolution or mixing of the water-insoluble compound.

7: The immersion mixture as claimed in claim 6, wherein the water-insoluble compound is an ethoxylated perfluoropolyether having terminal dialcohols or a 2,3,3,3-tetrafluoro-2-[1,1,2,3,3,3-hexafluoro-2-(heptafluoropropoxy)propoxy]-1-propanol.

8: The immersion mixture as claimed in claim 6, wherein the water-insoluble compound is an esterified perfluoropolyether of the chemical structure
R1-O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CF.sub.2O(CF.sub.2CF.sub.2O).sub.p(CF.sub.2O).sub.q CF.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.nO—R2 wherein the groups R1 and R2 are each either phenylacetate ester, tetrahydrofurancarboxylate ester, or (2-methoxyethoxy)acetate ester; n=1 to 3; and p and q are each chosen from within a range from 3 to 12.

9: The immersion mixture as claimed in claim 6, wherein the proportion of the respective water-soluble organic, water-soluble inorganic, or water-insoluble compounds is at least 30 percent by weight.

10: A method of microscopy, the method comprising: preparing the immersion mixture as claimed in claim 6 as an immersion medium.

11: The immersion mixture as claimed in claim 6, wherein the water-insoluble compound is a compound of the chemical structure ##STR00004## wherein the group R3 is a phenylacetate ester (radical).

Description

[0032] Examples of possible immersion mixtures and of the components thereof are given below.

[0033] The immersion mixtures may be divided into the following groups: [0034] mixtures of water and inorganic substances (salt solutions), [0035] mixtures of water and organic compounds, [0036] mixtures of high-boiling organic compounds that are water-soluble, and [0037] mixtures of water-insoluble organic compounds.

[0038] Combinations of one or more such mixtures are of course also possible in principle here, for example a mixture of water, inorganic substances, and organic compounds. Mixtures of water-soluble, organic compounds and water-insoluble compounds are possible too.

[0039] By combining organic components having different chemical structures, it is also possible to vary to some degree the dispersion of an immersion medium, as expressed by the Abbe number ve.

[0040] A: Mixtures of Water and Inorganic Substances

[0041] Mixtures of water and inorganic substances such as potassium carbonate or sodium citrate can be produced either by mixing a highly concentrated solution of the inorganic substance and water, for example in a mixer system. For example, high-concentration and low-concentration aqueous solutions of the salts can be mixed. It is also possible to add a defined amount of a pure salt in solid form to an aqueous or low-concentration solution and mix this together. The refractive index is determined or measured as an actual value, for example on the basis of theoretical models as a function of the concentration of one or more salts. To suppress colonization of the immersion mixture by fungi, bacteria, and/or algae and to prolong the shelf life, herbicides, algicides and/or bactericides may be added to the immersion mixture. If they have an influence on the resulting optical properties of the immersion mixture, these biocidal additives are also treated as components having different optical properties. A disadvantage of water-containing immersion mixtures is the possible evaporation of water fractions, which, in addition to the change in the refractive index during the measurement, can result in problems due to crystalline deposits. Mention should in particular be made here of the potential for clogging of the pumps and/or of the mixer system and for contamination of the sample space.

[0042] Examples of suitable substances for immersions based on aqueous solutions of inorganic substances are: [0043] ammonium sulfate, molecular weight 132.1 g/mol, solubility in water (20° C.) 754 g/L (Merck), aqueous solution 200 g/L (1.51 mol/L): ne=1.3629; [0044] sodium benzoate, molecular weight 144.1 g/mol, solubility in water (20° C.) 660 g/L (Merck), aqueous solution 200 g/L (1.39 mol/L): ne=1.3770; [0045] sodium chloride, molecular weight 58.4 g/mol, solubility in water (20° C.) 358 g/L (Merck), aqueous solution 200 g/L (3.42 mol/L): ne=1.3656; [0046] sodium salicylate, molecular weight 160.1 g/mol, solubility in water (20° C.) 1000 g/L (Merck), aqueous solution 200 g/L (1.25 mol/L): ne=1.3747; [0047] sodium thiocyanate (sodium rhodanide), molecular weight 81.1 g/mol, solubility in water (20° C.) 1200 g/L (Merck), aqueous solution 200 g/L (2.47 mol/L): ne=1.3765; [0048] sodium molybdate dihydrate, molecular weight 241.95 g/mol, solubility in water (20° C.) 840 g/L (Merck), aqueous solution 200 g/L (0.83 mol/L): ne=1.3619; [0049] sodium tungstate dihydrate, molecular weight 329.9 g/mol, solubility in water (20° C.) 730 g/L (Merck), aqueous solution 200 g/L (0.61 mol/L): ne=1.3531; and [0050] magnesium sulfate heptahydrate, molecular weight 246.5 g/mol, solubility in water (20° C.) 710 g/L (Merck), aqueous solution 200 g/L (0.81 mol/L): ne=1.3529.

[0051] B: Mixtures of Water and Organic Compounds

[0052] It is also possible to mix one or more organic compounds with water. It is likewise possible to prepare aqueous solutions (extracts) beforehand and mix them with one another. Alcohols (mono-, di-, and polyfunctional) are particularly suitable as organic compounds, as these usually have good miscibility with water. Examples include: aqueous solutions of ethylene glycol, propylene glycol, polyethylene glycols, polypropylene glycols, butanediols, tetrahydrofurfuryl alcohol, tetrahydrofurfuryl alcohol polyethylene glycol ether (glycofurol), 1,4-cyclohexanedimethanol, glycerol, trimethylolethane, glycol ethers, and polyacetals. Likewise advantageous can be the addition of tertiary amines, amides, polyacrylic acid and copolymers, polyacrylamides, polyvinyl alcohols, sugars and sugar alcohols, and citric acid. Using a suitable mixing unit (mixer system), various aqueous solutions can be used to adjust and mix the immersion mixture to a desired refractive index as the actual value, for example within a range from ne=1.35 to 1.42 inclusive. The formation of deposits is practically impossible here. However, water can continue to evaporate, resulting in fluctuating quality of the image data in protracted measurements if the evaporation losses are not compensated from time to time or continuously. Such compensation of evaporating fractions of the immersion mixture can be achieved through the above-described comparison of actual values and target values or by means of an appropriately controlled mixing unit. An advantage of water-containing immersion mixtures is the easy cleaning of mixing and metering systems by rinsing with water, in particular deionized water.

[0053] Examples of suitable substances for water-containing immersions are: [0054] tetrahydrofurfuryl alcohol polyethylene glycol ether (glycofurol), ne=1.4621; [0055] ethylene glycol diacetate, ne=1.4161; [0056] tetraoxaundecane, ne=1.4144; [0057] diethylacetoacetamide, ne=1.4728; [0058] 2,2′-thiodiethanol, ne=1.5236; [0059] triethanolamine, ne=1.4868; [0060] polypropylene glycol 400, ne=1.4480; [0061] dipropylene glycol, ne=1.4417; [0062] diethylene glycol diethyl ether, ne=1.4126; and [0063] dimethyl propylene urea, ne=1.4909.

[0064] C: Mixtures of High-Boiling Organic Substances that are Water-Soluble

[0065] In a further embodiment of the invention it is possible for the abovementioned compounds to be used directly as pure substances. In this case, two water-soluble pure substances are used in an immersion mixture and this is used as an immersion medium. Here too, it is again possible to adjust the refractive index of the immersion mixture. The advantage of this approach is that no deposits form here and, because of the high-boiling nature of the immersion components, there are no adverse evaporation effects. In addition, the immersion mixture can be easily removed with water. However, measurements in conditions of high atmospheric humidity can lead to the absorption of water by the immersion mixture, as a result of which the refractive index may change during the measurement. In addition to the compounds mentioned, glycol-terminated short-chain perfluoropolyethers are also suitable here, particularly in the form of a mixture with alcohols, diols, polyols or glycol ethers.

[0066] Examples of substances (fluorine-free) that are suitable for live-cell immersions (non-aqueous) are: [0067] methylphenyl acetate; ne(23)=1.5092; [0068] diethyl maleate; ne(23)=1.4414; [0069] diethyl fumarate; ne(23)=1.4419; [0070] dimethyl maleate; ne(23)=1.4435, [0071] bis (2-ethylhexyl) sebacate; ne(23)=1.4511, [0072] diethyl succinate; ne(23)=1.4200; [0073] dimethyl adipate; ne(23)=1.4289; [0074] triethylene glycol bis(2-ethylhexanoate); ne(23)=1.4449; [0075] triethyl citrate; ne(23)=1.4431; [0076] di-n-butyl carbonate; [0077] malondialdehyde bis(dimethyl acetal); ne(23)=1.4070; [0078] 2-ethylhexane-1,3-diol; ne(23)=1.4518; [0079] diacetone alcohol (4-hydroxy-4-methyl-2-pentanone); ne(23)=1.4244; [0080] 2-ethylhexyl phosphate, mixture of mono- and diesters; ne(23)=1.4437; and [0081] diethylacetamide; ne(23)=1.4408.

[0082] D: Mixtures of Water-Insoluble Organic Compounds

[0083] High-boiling, water-insoluble compounds may likewise be used in multi-component immersion mixtures that are mixed directly in situ. Functional fluorinated organic compounds are particularly suitable here, since, in addition to a very low refractive index, they also have very high dispersion. This means that admixing of just small amounts may be sufficient to achieve large optical effects in respect of refractive index and dispersion. The chemical modification of organofluorine compounds having functional end or side groups such as glycol, alkyl ether, carboxyl, ester, vinyl, allyl, acryloyl/methacryloyl or alkyl makes it possible to mix these with non-fluorinated substances. Pure perfluoro compounds are, on the other hand, absolutely incompatible with other organic substances. In addition, these modifications can at the same time serve to adapt the refractive index. For example, the refractive index is increased by esterification with aromatic carboxylic acids, whereas the dispersion decreases. By mixing a plurality of organofluorine and/or aliphatic, cycloaliphatic esters and ethers, the resulting refractive index of the immersion mixture can likewise be adapted to the application in situ.

[0084] In one embodiment of the immersion mixture according to the invention, the water-insoluble compound is an ethoxylated perfluoropolyether having terminal dialcohols or a 2,3,3,3-tetrafluoro-2-[1,1,2,3,3,3-hexafluoro-2-(heptafluoropropoxy)propoxy]-1-propanol.

[0085] Examples of such fluorine compounds are: [0086] Fluorolink® E10-H* (bifunctional glycol-terminated perfluoropolyether), molecular weight approx. 1500 g/mol, ne=1.3180; [0087] Fluorolink® D10* (alcohol-terminated perfluoropolyether); [0088] Fluorolink® L10* (perfluoropolyether dicarboxylic acid dimethyl ester); [0089] Cheminox FA-6; 2-(perfluorohexyl)-ethanol; [0090] Cheminox PO-3-ME; methyl 2,3,3,3-tetrafluoro-2-[1,1,2,3,3,3-hexafluoro-2-(heptafluoropropyl)-propoxy] propanoate; [0091] Cheminox PO-3-OH; 2,3,3,3-tetrafluoro-2-[1,1,2,3,3,3-hexafluoro-2-(1,1,2,3,3,3-heptafluoropropoxy)propoxy]-1-propanol, ne=1.2944; and [0092] Cheminox OXF-3PO-OH; bis(hydroxymethyl)perfluoropolyether, MW=536 g/mol, ne=1.3220. [0093] Fluorolink® E10-H is a perfluoropolyether of the formula

O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CF.sub.2O(CF.sub.2CF.sub.2O).sub.p(CF.sub.2O).sub.qCF.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.nO.

[0094] Chominox PO-3-OH is a compound having the structural formula:

##STR00001##

[0095] The above structural formula is shown in two modes of representation.

[0096] Cheminox PO-3-OH is also known in the chemical nomenclature as 2,3,3,3-tetrafluoro-2-[1,1,2,3,3,3-hexafluoro-2-(heptafluoropropoxy)propoxy]-1-propanol (product list; HFPO derivatives; Unimatec).

[0097] In the course of this invention, new chemical compounds have been found, synthesized, and used as components in immersion mixtures of the invention.

[0098] One of these new chemical compounds is an esterified perfluoropolyether of the chemical structure

R1-O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CF.sub.2O(CF.sub.2CF.sub.2O).sub.p(CF.sub.2O).sub.qCF.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.nO—R2,

[0099] where the groups R1 and R2 are each either [0100] phenylacetate ester, [0101] tetrahydrofurancarboxylate ester or [0102] (2-methoxyethoxy)acetate ester

[0103] In these structures, n=1-3. The indices p and q are each within a range from 3 to 12. The advantageous effect of the groups R1 and R2 consists inter alia of a lowering of the refractive index (v value) due to the cyclic structures at the ends (phenylacetate ester and tetrahydrofurancarboxylate ester) and also that of a solubilizer (especially (2-methoxyethoxy)acetate ester) for the generally sparingly soluble fluorinated compounds.

[0104] Examples of these esterified perfluoropolyethers are: [0105] Fluorolink E10 bis(phenylacetate ester); ne=1.3697; [0106] Fluorolink E10 bis(tetrahydrofurancarboxylate ester); ne=1.3480; and [0107] Fluorolink E10 bis[(2-methoxyethoxy)acetate ester]; ne=1.3461.

[0108] The synthesis of the compound Fluorolink E10 bis(phenylacetate ester) can in one embodiment be carried out via a free acid. This is done by dissolving 150 g of Fluorolink E10 (0.1 mol) together with 2.2 equivalents of phenylacetic acid, with heating, in 100 ml of methyl isobutyl ketone and adding 0.95 g of para-toluenesulfonic acid. 3.6 ml of water of reaction collects in the water separator within 3 h (quant.). The reaction solution is washed with three 50 ml portions of water, then with one 50 ml portion of 10% K.sub.2CO.sub.3 solution, and finally with two more 50 ml portions of water (pH 7-8). The solvent is removed and the ester is obtained.

[0109] The compound Fluorolink E10 bis(phenylacetate ester) can in a further embodiment be synthesized via an acid chloride. This is done by dissolving 150 g of Fluorolink E10 together with 25 g of N-methylpiperidine, with heating, in 100 ml of methyl isobutyl ketone followed by dropwise addition of 2.4 equivalents of phenylacetyl chloride. After one hour under reflux, the reaction is complete. The reaction solution is washed with three 50 ml portions of water. The solvent is removed and the ester is obtained.

[0110] The compounds Fluorolink E10 bis(tetrahydrofurancarboxylate ester) and Fluorolink E10 bis[(2-methoxyethoxy)acetate ester] can be synthesized via a free acid.

[0111] 300 g of Fluorolink E10 (0.2 mol) is dissolved together with either 2.2 equivalents of tetrahydrofuran-2-carboxylic acid or 2.2 equivalents of 2-(2-methoxyethoxy)acetic acid, with heating, in 200 ml of methyl isobutyl ketone and 2 g of para-toluenesulfonic acid is added. 7.2 ml of water of reaction collects in the water separator within 3 h (quant.). The reaction solution is washed with four 50 ml portions of water. The solvent is removed and the ester is obtained after filtration through activated charcoal.

[0112] Another new chemical compound has the following chemical structure:

##STR00002##

[0113] The above structural formula is shown in two modes of representation. The group R3 is a phenylacetate ester (radical).

[0114] An example of such a chemical compound is Cheminox PO-3-OH phenylacetate ester; ne=1.3551.

[0115] The compound Cheminox PO-3-OH phenylacetate ester can be synthesized via a free acid. This is done by dissolving Cheminox PO-3-OH together with 2.2 equivalents of phenylacetic acid, with heating, in toluene and adding para-toluenesulfonic acid. Water of reaction collects in the water separator (quant.). The reaction solution is washed with several portions of water. The solvent is removed and the ester is obtained.

[0116] Listed below are application examples for immersion mixtures that are particularly suitable for use as immersion media in live-cell imaging.

[0117] i) Examples for aqueous immersion mixtures IM 1/1 to IM 1/10 are (parts by wt.=parts by weight): [0118] IM 1/1: 66.75 parts by wt. water, [0119] 33.75 parts by wt. glycofurol, [0120] ne=1.3800, [0121] Dispersion ve=55.1, [0122] Viscosity (23° C.)=3.21 mm.sup.2/s, [0123] Surface tension against air (20° C.): 42.3 mN/m; [0124] IM 1/2: 74.8 parts by wt. water, [0125] 25.2 parts by wt. polypropylene glycol 400, [0126] ne=1.3700, [0127] Dispersion ve=52.9, [0128] Viscosity (23° C.)=3.6 mm.sup.2/s; [0129] IM 1/3: 67.5 parts by wt. water, [0130] 32.5 parts by wt. polypropylene glycol 400, [0131] 0.1 parts by wt. p-chloro-m-cresol (Preventol CMK), biocide, [0132] ne=1.3800, [0133] Dispersion ve=55.1, [0134] Viscosity (23° C.)=5.3 mm.sup.2/s; [0135] IM 1/4: 65.9 parts by wt. water, [0136] 25.6 parts by wt. diethylacetoacetamide, [0137] 8.5 parts by wt. tetraoxaundecane, [0138] ne=1.3800, [0139] Dispersion ve=50.7, [0140] Viscosity (23° C.)=2.7 mm.sup.2/s; [0141] IM 1/5: 80.85 parts by wt. water, [0142] 19.15 parts by wt. glycofurol, [0143] ne=1.3600, [0144] Dispersion ve=55.4, [0145] Viscosity (23° C.)=1.86 mm.sup.2/s, [0146] Surface tension against air (20° C.): 40.2 mN/m; [0147] IM 1/6: 75.2 parts by wt. water, [0148] 24.8 parts by wt. dipropylene glycol, [0149] ne=1.3650, [0150] Dispersion ve=57.9, [0151] Viscosity (23° C.)=2.5 mm.sup.2/s, [0152] Surface tension against air (20° C.): 46.6 mN/m; [0153] IM 1/7: 77.85 parts by wt. water, [0154] 22.15 parts by wt. polypropylene glycol 400, [0155] ne=1.3650, [0156] Dispersion ve=55.3, [0157] Viscosity (23° C.)=3.0 mm.sup.2/s, [0158] Surface tension against air (20° C.): 39.5 mN/m; [0159] IM 1/8: 81.5 parts by wt. water, [0160] 18.5 parts by wt. 2,2′-thiodiethanol, [0161] ne=1.3650, [0162] Dispersion ve=52.1, [0163] Viscosity (23° C.)=1.7 mm.sup.2/s, [0164] Surface tension against air (20° C.): 52.4 mN/m; [0165] IM 1/9: 73.8 parts by wt. water, [0166] 26.2 parts by wt. diethylene glycol diethyl ether, [0167] ne=1.3650, [0168] Dispersion ve=55.3, [0169] Viscosity (23° C.)=2.6 mm.sup.2/s, [0170] Surface tension against air (20° C.): 35.95 mN/m; [0171] IM 1/10: 80.9 parts by wt. water, [0172] 19.1 parts by wt. N,N′-dimethylpropyleneurea, [0173] ne=1.3650, [0174] Dispersion ve=50.7, [0175] Viscosity (23° C.)=1.9 mm.sup.2/s, [0176] Surface tension against air (20° C.): 45.5 mN/m.

[0177] ii) Examples for anhydrous immersion mixtures IM 2/1 to IM 2/4 are: [0178] IM 2/1: 70.65 parts by wt. Fluorolink E10 H, [0179] 29.35 parts by wt. diethylacetoacetamide, [0180] ne=1.3800, [0181] Dispersion ve=52.8, [0182] Viscosity (23° C.)=45 mm.sup.2/s; [0183] IM 2/2: 69.55 parts by wt. malondialdehyde bis(dimethylacetal), [0184] 30.45 parts by wt. Cheminox PO-3-OH, [0185] ne=1.3800, [0186] Dispersion ve=65.5, [0187] Viscosity (23° C.)=2.0 mm.sup.2/s; [0188] IM 2/3: 48.8 parts by wt. diethyl fumarate, [0189] 51.2 parts by wt. Cheminox PO-3-OH, [0190] ne=1.3800, [0191] Dispersion ve=47.5, [0192] Viscosity (23° C.)=3.0 mm.sup.2/s; [0193] IM 2/4: 66.6 parts by wt. Cheminox OXF-3PO-OH, [0194] 34.4 parts by wt. dimethyl adipate, [0195] ne=1.3650, [0196] Dispersion ve=71.6, [0197] Viscosity (23° C.)=9.6 mm.sup.2/s.

[0198] iii) Examples for immersion mixtures IM 3/1 to IM 3/4 comprising water and at least one inorganic component (salt solutions) are: [0199] IM 3/1: Magnesium sulfate heptahydrate solution in water, [0200] 345 g/L or 302 g/kg, [0201] ne=1.3650, [0202] Dispersion ve=57.9; [0203] IM 3/2: Sodium chloride solution in water, [0204] 194 g/L or 178 g/kg, [0205] ne=1.3650, [0206] Dispersion ve=52.1; [0207] IM 3/3: Ammonium sulfate solution in water, [0208] 175 g/L or 165 g/kg, [0209] ne=1.3600, [0210] Dispersion ve=57.1; [0211] IM 3/4: Sodium benzoate solution in water, [0212] 190 g/L or 181 g/kg, [0213] ne=1.3750, [0214] Dispersion ve=47.5.

[0215] All of the abovementioned examples of the immersion mixture according to the invention advantageously contain the respective compounds in a proportion of at least 30 percent by weight (parts by wt.=wt %) in addition to the water, where present.