GB2419427A

GB2419427A - Mountant solution for a microscope

Info

Publication number: GB2419427A
Application number: GB0521095A
Authority: GB
Inventors: Robert Stephen Davidson
Original assignee: Individual
Current assignee: Individual
Priority date: 2004-10-19
Filing date: 2005-10-17
Publication date: 2006-04-26
Anticipated expiration: 2025-10-17
Also published as: GB0423182D0; GB2419427B; GB2419425A; GB0521095D0

Abstract

A microscope has a microscope slide supporting a biological specimen mounted in a mountant solution. The same solution acts as an immersion oil between the slide and a lens of the microscope. The solution has a refractive index within 5% of that of the lens and the slide and optionally that of a cover slip. A liquid mountant solution comprises a water miscible polyol or polymer or a buffer solution, and sufficient water soluble additives to increase the refractive index of the solution to 1.50 - 1.75 measured at 22{ on the sodium D line.

Description

241 9427

MOUNTANT SOLUTIONS

The present invention relates to solutions which can be used both as immersion oils and as mountant solutions, i.e. solutions used for mounting biological specimens for viewing e.g. with a microscope.

Immersion oils are liquids having a refractive index which matches that of the glass used in the optics of a microscope and the cover slip which covers the specimen mounted on a microscope slide which is held on the microscope stage. When light travels between media, it is refracted at each interface and hence, during microscopy using a conventional transmission microscope, the light is refracted at the cover slip - air interface and at the air - microscope objective interface. For work requiring high resolution, an immersion oil is used between the lens of the microscope and the slide holding the specimen, so as to minimise refraction and hence distortion of the image. A similar situation obtains with fluorescence microscopy where the excitation light is channelled via the microscope optics so as to impinge upon the specimen and the emitted light travels back through the microscope optics. The growth of confocal microscopy where a three dimensional image of the specimen is built up is another technique where it is important to avoid distortion due to refraction. Another technique where it is important to eliminate refraction effects is multiphoton microscopy, which is becoming of increasing interest.

In the early days, immersion oils were natural oils such as cedar wood oil or halogenated aromatic compounds such as iodonaphthalene etc. More recently oils, which are halogen-free, have been introduced such as the Cargille range of oils and others which are marketed by microscope suppliers (Zeiss, Olympus etc.). All these oils have a property in common: they are water immiscible.

Even though an immersion oil may be used, refraction can also occur as light passes through the medium in which the specimen is mounted. The current range of immersion oils cannot be used as mounting media as they are water immiscible and hence do not wet the biological specimen.

Solutions commonly used as mountant solutions include aqueous solutions of polyols, such as glycerol, or aqueous solutions of polymers, such as polyvinyl alcohol.

Often these solutions include buffers, such as phosphate buffered saline or trig-buffer, to achieve a desired pH value. However, these solutions have refractive indices which differ from those of the glass of which most microscope lens are made, and so refraction and distortion of the image are inevitable.

I have now discovered a range of solutions which can be used both as immersion oils and as mountant solutions.

Thus, in one aspect, the present invention consists in a microscope including a prepared glass microscope slide, where the microscope slide supports a biological specimen mounted in a mountant solution, the microscope having the same mountant solution between said slide and a lens of the microscope, the mountant solution having a refractive index within 5% of the refractive index of the lens and of the slide.

When a refractive index is referred to herein, it is always as measured at 22 C on the sodium D line, unless noted otherwise.

In a further aspect, the present invention consists in a liquid mountant solution comprising a water-miscible, preferably water-soluble, polyol or polymer or a buffer solution, and sufficient water-soluble additives to increase the refractive index of the solution to a value from 1.50 to 1. 75, measured at 22 C on the sodium D line.

In a further aspect, the present invention consists in a prepared microscope slide supporting a biological specimen mounted in a mountant solution of the present invention, and covered with a cover slip, the refractive index of the mountant solution being within 5%, more preferably within 3% and most preferably within 2%, of the refractive index of the cover slip.

In a still further aspect, the invention provides a microscope supporting a prepared glass microscope slide according to the present invention and having a mountant solution of the present invention between said slide and a lens of the microscope, the mountant solution having a refractive index within 5%, more preferably within 3% and most preferably within 2%, of the refractive index of the lens.

The mountant solution of the present invention, when it is intended for use for mounting biological specimens, is preferably hydrophilic, or, at least, water-compatible.

The majority of glasses used in microscope lenses and slides have a refractive index of about 1.52, most commonly about 1.515, and so the preferred solutions of the present invention have a refractive index close to this value, e.g. from 1.51 to 1.53.

However, a wider range of values is possible, depending on the glass used, for example from 1.505 to 1.570.

Examples of polyols which may be employed in the present invention include: glycerol, ethoxylated pentaerythritol, ethoxylated trimethylolpropane, triethylene glycol, pentaerythritol and trimethylolpropane. Examples of suitable water-miscible polymers include: polyethylene glycol, polyvinyl alcohol, poly(vinylpyrrolidone), poly(hydroxyalkyl acrylates) [e.g. poly(2-hydroxyethyl acrylate)], polyacrylamides; phthalate esters, such as poly[(diethylene glycol) phthalate]diol; and other hydroxy terminated linear polymers e.g. polyethers or polycarbonates.

Where the polyol or polymer is normally solid, it is dissolved in a suitable solvent, normally and preferably water.

The amount of the polyol and/or polymer employed will depend on the solution viscosity which it is desired to achieve. However, in general, an amount close to saturation is preferred.

Where a buffer is used, this is preferably buffered to a pH which is the most appropriate for the fluorochrome used as the biological marker (usually between pH 7 and 10). One suitable buffer solution is a glycerolphosphate buffered saline solution, such as Citifluor AFl solution or trig-buffer.

Additives which may be used to increase the refractive index of the solution of the present invention include methyl phenyl sulphoxide, pyridine N-oxide, aromatic esters, especially benzoate or phthalate esters and derivatives thereof, for example benzyl benzoate or benzyl 4methoxybenzoate. All of these compounds are soluble in or miscible with aqueous solutions of glycerol, polyvinyl alcohol, or various other polyols, such as triethylene glycol, diethylene glycol, ethoxylated pentaerythritol or ethoxylated trimethylolpropane. Other compounds which may be used include quinoline N-oxides, isoquinoline N-oxides and phosphine oxides.

The refractive indices of various types of glass are shown in the following Table 1:

Table 1

Type of glass Refractive index on Na D line Zinc crown 1.517 High dispersion crown 1.520 Light flint 1.575 Heavy flint 1.650 Heaviest flint 1.890 The invention is further illustrated by the following non- limiting Examples.

EXAMPLE 1

Solutions based on methyl phenvlsulphoxide and glycerol The following solutions were prepared from methyl phenylsulphoxide plus glycerol only.

Sulphoxide Glycerol nD 2g. 0.4g 1.552 v 2g lg 1.538 2g 2.4g 1.521

EXAMPLE 2

Methyl Phenvlsulphoxide plus Citifluor AF1 solution 1 g of methyl phenylsulphoxide plus 1.2g of Citifluor AF 1 solution (a glycerol phosphate buffered saline solution containing antifadent) gave a solution having nD= 1.515. The solution contained 5.45% water.

EXAMPLE 3

Methyl Phenvlsulphoxide plus Citifluor CFPVOH 2.5g of methyl phenylsulphoxide plus 1 g of Citifluor CFPVOH (an aqueous solution of poly(vinyl alcohol) gave a solution having nD = 1.513. The solution contained 22.86% water.

An advantage of this solution is that on evaporation of water a solid film is formed.

EXAMPLE 4

Methyl Phenvlsulphoxide plus phosphate buffered saline solution 1.2g of methyl Phenvlsulphoxide plus 0.4g of phosphate buffered saline gave a solution having nD of 1.505. The solution contained 25% water.

EXAMPLE 5

Methyl Phenvlsulphoxide plus aqueous poly(vinvl ovrrolidone) solution 1 g of methyl Phenvlsulphoxide plus 0.4g poly(vinyl pyrrolidone) solution gave a solution having nD = 1.514. The solution contained 22.86% water.

EXAMPLE 6

Methyl phenvlsulphoxide/Polvethvlene aIycol dibenzoate/elvcerol/triethvlene

PIYCOI

A quaternary mixture of lg of methyl phenylsulphoxide plus 0.5g polyethylene glycol dibenzoate plus 0.8g glycerol plus 0.2g triethylene glycol gave a solution having nD = 1.517. this solution contained no water.

EXAMPLE 7

Pvridine N-oxide plus aIvcerol Pyridine N-oxide Glycerol nD lg 0.5g 1.555 lg 1.5g 1.5225 10_._.

lg 1.8g 1.518 All of these solutions contain no water. For the most common lens glasses, the solution to be used would be that containing lg of pyridine N-oxide and 1.8g of glycerol.

EXAMPLE 8

Pvridine N-oxide plus Citifluor AF1 solution Pyridine N-oxide AF1 solution nD lg 1.2g 1.525 lg 1.55g 1.515 For the most common lens glasses, the solution to be used would be that containing lg pyridine N-oxide plus 1.55g AF1 solution. This solution contains 6.08% water.

EXAMPLE 9

Pvridine N-oxide plus aqueous phosphate buffered saline (PBS) solution Pyridine N-oxide PBS solution nD lg 0.3g 1.53 lg 0.4g 1.518 For the most common lens glasses, the solution to be used would be that containing lg pyridine N-oxide plus 0.4g PBS solution. This solution contains 28.57% water.

EXAMPLE 10

Pyridine N-oxide plus CFPVOH [aqueous poly(vinYI alcohol) solution! 1 g of the N-oxide plus 0.35g of CFPVOH had an nD of 1.517 and contained 20.74% water.

EXAMPLE 11

Pvridine N-oxide plus PVP [aqueous poly(vinvl ovrrolidone) solution! lg pyridine N-oxide plus 0.5g PVP solution had an nD of 1.5134 and contained 26.67% water

EXAMPLE 12

Poly(ethvlene t!lvcol) dibenzoate (PEG dibenzoate) plus ethoxylated pentserythritol (PP 150) 5.6g of PEG dibenzoate plus l.5g PP150 had an nD of 1.515.

EXAMPLE 13

PEG dibenzoate plus ethoxvlated trimethvlolpropane (TP 70) 5.6g of PEG dibenzoate plus 1.5g TP70 had an nD of 1.5125.

EXAMPLE 14

pEG dibenzoate plus ethexylated pentaerythritol (TP 200) 5.6g of PEG dibenzoate plus l.5g TP200 had an nD of 1.5135.

EXAMPLE 15

PEG dibenzoate plus triethylene aIycol 5.6g of PEG dibenzoate plus l.5g triethylene glycol had an nD of 1.5105.

EXAMPLE 16

Polvl(diethYlene zIycol)phthalateldiol plus PEG dibenzoate plus triethylene aIycol 4.29g of the polyester plus 2.23g PEG dibenzoate plus 1 g triethylene glycol had an nD of 1.517.

EXAMPLE 17

Quinoline N-oxide (hydrate) plus aIvcerol j qumolineN-oxide glycerol nD lg I lg 1.5385 lg 1 1.56g 1.5235 lg 1 1.81g 1 1.5190

EXAMPLE 18

Quinoline N-oxide (hydrate) plus AF1 solution lg quinoline N-oxide plus l. 55g AF1 gave an nD 1.5170 and contained 6.1% water.

EXAMPLE 19

4-Picoline N-oxide plus aIvcerol Ig N-oxide plus 1.46g glycerol gave an nD 1.5215.

EXAMPLE 20

4-Picoline N-oxide plus AF1 solution lg N-oxide plus 1.39g AFl solution gave an nD 1.5185 and contained6.03% water.

EXAMPLE 21

2-Picoline N-oxide plus Glycerol 2-Picoline N-oxide Glycerol nD lg lg 1.5310 lg 1.4g 1.5200 1

EXAMPLE 22

2-Picoline N-oxide plus AF1 2-Picoline N-oxide AF1 nD lg lg 1.5250 lg 1.25g 1.5190 For the most common lens glasses, the solution to be used would be that containing lg picoline N-oxide plus 1.25g AFT. This solution contains 6.03% water.

EXAMPLE 23

26-Lutidine N-oxide plus Glycerol 2,6-Lutidine N-oxide Glycerol nD lg lg 1.5230 lg 1.33g 1.5150

EXAMPLE 24

26-Lutidine N-oxide plus AF1 lg N-oxide pluslg AF1 gave nD 1.5165 and contained 6.03% water.

EXAMPLE 25

Benzyl Benzoate Polyester (4.06g) was mixed with benzyl Benzoate (2.04g) to give a solution having a refractive index of 1.539.

EXAMPLE 26

Benzvl Benzoate plus Triethylene Glycol The solution of Example 25 (l.Og) was mixed with triethylene glycol containing 10% water (0.25g) to give a solution having refractive index of 1.515.

EXAMPLE 27

Benzyl Benzoate plus Triethylene Glycol The solution of Example 25 (2.0g) was mixed with triethylene glycol (0.5g) to give a solution having a refractive index of 1.5185.

EXAMPLE 28

Benzyl Benzoate Polyester (4.62g) was mixed with benzyl Benzoate (4.61g) to give a solution having a refractive index of 1.5465.

EXAMPLE 29

Benzyl Benzoate plus Diethylene GIYCOI The solution of Example 25 (I.Og) was mixed with diethylene glycol (0.25g) to give a solution having a refractive index of 1.5165

EXAMPLE 30

Benzyl Benzoate A solution of 1,4-diazabicylo[2,2,2]octane (DABCO)(1.25g) in triethylene glycol(lOg) and water (la) was made up. 0.25g ofthis solution was added to the solution of Example 25 (1 g) to give a solution having a refractive index of 1.519. 1,4- diazabicylo[2,2,2]octane is an antifadent and is incorporated into the mountant solution to retard the fading of fluorochromes.

Claims

CLAIMS: 1. A microscope including a prepared glass microscope slide, where

the microscope slide supports a biological specimen mounted in a mountant solution, the microscope having the same mountant solution between said slide and a lens of the microscope, the mountant solution having a refractive index within 5% of the refractive index of the lens and of the slide.
2. A microscope according to Claim 1, in which the mountant solution is hydrophilic.
3. A microscope according to Claim 2, where the mountant solution comprises a water- miscible polyol or polymer.
4. A microscope according to Claim 3, in which the polyol is glycerol, ethoxylated pentaerythritol, ethoxylated trimethylolpropane, triethylene glycol, pentaerythritol or trimethylolpropane.
5. A microscope according to Claim 3, in which the water-miscible polymer is polyethylene glycol, polyvinyl alcohol, poly(vinylpyrrolidone), poly(hydroxyalkyl acrylates), poly(2-hydroxyethyl acrylate), a polyacrylamide or poly[(diethylene glycol)phthalate] diol.
6. A microscope according to any one of Claims 3 to 5, in which said polyol or polymer is solid and employed in the form of a solution.
7. A microscope according to Claim 6, in which the solution is a solution in water.
8. A microscope according to Claim 1 or Claim 2, where the mountant solution comprises a buffer solution.
9. A microscope according to Claim 8, in which the buffer solution is a saline- containing buffer solution.
l O. A microscope according to any one of the preceding Claims, in which the slide and lens each has a refractive index in the range from 1.51 to 1.53.
11. A microscope according to any one of the preceding Claims, in which the mountant solution has a refractive index in the range from 1.51 to 1. 53.
12. A microscope according to any one of the preceding Claims, in which the mountant solution includes an additive selected from methyl phenyl sulphoxide, pyridine N-oxide, a quinoline N-oxide, an isoquinoline Noxide or a phosphine oxide.
13. A liquid mountant solution comprising a water-miscible polyol or polymer or a buffer solution, and sufficient water-soluble additives to increase the refractive index of the solution to a value from 1.50 to 1. 75, measured at 22 C on the sodium D line.
14. A mountant solution according to Claim 13, in which the polyol is glycerol, ethoxylated pentaerythritol, ethoxylated trimethylolpropane, triethylene glycol, pentaerythritol or trimethylolpropane.
15. A mountant solution according to Claim 13, in which the watermiscible polymer is polyethylene glycol, polyvinyl alcohol, poly(vinylpyrrolidone), poly(hydroxyalkyl acrylates), poly(2-hydroxyethyl acrylate), a polyacrylamide or poly[(diethylene glycol)phthalate] diol.
16. A mountant solution according to any one of Claims 13 to 15, in which said polyol or polymer is solid and employed in the form of a solution.
17. A mountant solution according to Claim 16, in which the solution is a solution in water.
18. A mountant solution according to any one of Claims 13 to 17, additionally comprising an additive selected from methyl phenyl sulphoxide, pyridine N-oxide, a quinoline N-oxide, an isoquinoline Noxide or a phosphine oxide.
l 9. A prepared microscope slide supporting a biological specimen mounted in a mountant solution according to any one of Claims 13 to 18, and covered with a glass cover slip, the refractive index of the mountant solution being within 5% of the refractive index of the cover slip.
20. A microscope supporting a prepared microscope slide according to Claim 19, and having a mountant solution according to any one of Claims 13 to 18 between said slide and a lens of the microscope, the mountant solution having a refractive index within 5% of the refractive index of the lens.