CONTRAST SOLUTION FOR THE CHARACTERISATION OF BIOLOGICAL SAMPLES BY ELECTRON OR CORRELATIVE MICROSCOPY

Abstract

The invention relates to a solution comprising: a heteropolyacid and a lanthanide salt in a solvent consisting of a water/organic solvent mixture or an organic solvent. Optionally, the solution can also comprise an organic or inorganic buffer with a pH comprised in the interval of 4-6 or a strong base or acid. The object of the present invention also includes a use of said solution and any products of the reaction between the components thereof as a contrast medium for biological samples and a method for preparing biological samples that uses said solution in at least one step, for analysis by either correlative microscopy (CLEM) and electron microscopy (EM).

Claims

1. A solution comprising: a) a heteropolyacid with the general formula H.sub.3PM.sub.12O.sub.40, wherein M is tungsten or molybdenum, b) a lanthanide salt with the general formula Ln(III)A.sub.x*nH.sub.2O, wherein Ln is an element belonging to the series of lanthanides other than promethium, A is an organic or inorganic anion, x is a whole number greater than or equal to 1, and n is a whole number greater than or equal to 0; c) a solvent consisting of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is selected from a ketone and an alcohol and, in the case of a water/organic solvent mixture, is present in an amount comprised from 1 to 99% (v/v) relative to the total volume of the mixture in the case of ketone, or in an amount comprised from 71 to 99% (v/v) relative to the total volume of the mixture in the case of alcohol.

2. The solution according to claim 1, wherein the molar ratio between the component a) and the component b) (b/a molar ratio) is comprised in the interval of 0.1-100.

3. The solution according to claim 1, wherein the concentration of the component b) in the solvent c) is comprised from 0.01 to 250 mM.

4. The solution according to claim 1, wherein the component b) has an element selected from ytterbium, europium, terbium and gadolinium as a cation.

5. The solution according to claim 1, wherein the component b) has a halide as an anion.

6. The solution according to claim 1, wherein the solvent c) consists of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is selected from a ketone and a alcohol and is present, in the case of the water/organic solvent mixture, in an amount comprised from 20 to 99% (v/v), relative to the total volume of the mixture in the case of ketone, or in an amount comprised from 80 to 95% (v/v), relative to the total volume of the mixture in the case of alcohol.

7. The solution according to claim 1, wherein the solvent c) consists of a water/organic solvent mixture in which said organic solvent is a ketone with a number of carbon atoms comprised from 1 to 4, or an alcohol with a number of carbon atoms comprised from 1 to 4.

8. The solution according to claim 1, wherein the solvent c) consists of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is selected from acetone, methanol and ethanol.

9. A method for the characterisation of biological samples in suspension (negative contrast) or embedded in resin by means of correlative microscopy (CLEM) or electron microscopy (EM) with the solution according to claim 1 as a contrast medium.

10. The method according to claim 9, with the compound obtained from the reaction between the components a) and b) according to claim 1 under ambient pressure and temperature conditions and at a pH comprised in the interval of 4-6, wherein said compound has the formula [YbPW.sub.9O.sub.34].sup.6−.

11. A method for preparing biological samples for analysis by correlative microscopy (CLEM) or electron microscopy (EM), comprising the following steps: i) performing a physical fixation of the biological sample to be analysed, following the high pressure freezing (HPF) method; ii) performing the technique of substituting water with an organic solvent at cryogenic temperatures (FS—freeze substitution) in a frozen biological sample obtained in step (i), where the substitute medium is the solution comprising the components a) to c) according to claim 1.

12. The method according to claim 11, wherein said solution as per step ii) is obtained starting from an intermediate solution comprising the components a) and b) in a solvent c′), said solvent c′) being an aqueous or hydroalcoholic solvent containing up to 70% (v/v) alcohol, said intermediate solution then being dried and subsequently reconstituted in the solvent c).

13. The solution according to claim 1 wherein the molar ratio between the component a) and the component b) (b/a molar ratio) is comprised in the interval of 1-50.

14. The solution according to claim 1 wherein the molar ratio between the component a) and the component b) (b/a molar ratio) is comprised in the interval of 1-15.

15. The solution according to claim 1, wherein the concentration of the component b) in the solvent c) is comprised from 0.1 to 100 mM.

16. The solution according to claim 1, wherein the concentration of the component b) in the solvent c) is comprised from 1 to 50 mM.

17. The solution according to claim 1, wherein the solvent c) consists of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is selected from a ketone and a alcohol and is present, in the case of the water/organic solvent mixture, in an amount comprised from 90 to 95% (v/v), relative to the total volume of the mixture in the case of ketone, or in an amount comprised from 90 to 95% (v/v) relative to the total volume of the mixture in the case of alcohol.

18. The solution according to claim 1, wherein the solvent c) consists of a water/organic solvent mixture in which said organic solvent is a ketone with a number of carbon atoms comprised from 1 to 3, or an alcohol with a number of carbon atoms comprised from 1 to 3.

19. The solution according to claim 1, wherein the solvent c) consists of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is acetone.

Description

DESCRIPTION OF THE FIGURES

[0022] FIG. 1 shows the parameters used by an automated embedder in the protocol for substituting water with an organic solvent at cryogenic temperatures (FS—Freeze Substitution), used for preparing biological samples to be characterised by means of the CLEM technique according to example 2.

[0023] FIG. 2 shows confocal microscope images for comparing cells prepared with different contrast protocols according to example 2. The substitution (FS) medium used is: 100% acetone, “Unstained” (a); 0.1% uranyl acetate in acetone, “UA” (b); a stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/10 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/10” (c); a stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/16 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/16” (d); and a stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/30 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/30” (e).

[0024] FIG. 3 shows images, acquired by means of the transmission electron microscopy (TEM) technique, of the same cells observed by fluorescence microscopy as per example 2. The substitution (FS) medium used is: 100% acetone, “Unstained” (a); 0.1% of uranyl acetate in acetone, “UA” (b); a stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/10 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/10” (c); a stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/16 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/16” (d); and a stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/30 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/30” (e).

[0025] FIG. 4 shows representative images, acquired by means of the transmission electron microscopy (TEM) technique, of the cells shown in FIGS. 2 and 3, at a higher magnification. The substitution (FS) medium is the preferred medium according to the present invention, i.e. the stock solution obtained as per example 1, freeze-dried and reconstituted in acetone in a 1/10 (v/v) ratio relative to the volume of the solution prior to freeze-drying, “X Sol 1/10”.

[0026] FIG. 5 shows the CLEM procedure applied on a sample (Drosophila oocyte) prepared with the technique described in example 2 using ethanol as the organic solvent in the place of acetone, as per example 3.

DETAILED DESCRIPTION

[0027] “Conventional electron microscopy”, means microscopy that does not exploit light as the source of radiation, but rather an electron beam. Examples of “conventional electron microscopy” are scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The term “conventional electron microscopy” is thus used, for the purposes of the present invention, as a synonym of “electron microscopy (EM)” according to the definition given above.

[0028] “Organic solvent”, for the purposes of the present invention, means any organic solvent having a degree of purity, relative to the content of organic and inorganic compounds, comprised between 70 and 100% (v/v) (so-called “absolute”), and having a water content comprised between 50 and 0% (v/v) (so-called “anhydrous”).

[0029] In a first aspect, the present invention relates to a solution comprising: [0030] a) a heteropolyacid with the general formula H.sub.3PM.sub.12O.sub.40, wherein M is tungsten or molybdenum; [0031] b) a lanthanide salt with the general formula Ln(III)A.sub.x*nH.sub.2O, wherein Ln is an element belonging to the series of lanthanides other than promethium, A is an organic or inorganic anion, x is a whole number greater than or equal to 1, and n is a whole number greater than or equal to 0; [0032] c) a solvent consisting of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is selected from a ketone and an alcohol and, in the case of a water/organic solvent mixture, is present in an amount comprised from 1 to 99% (v/v), preferably from 20 to 99% (v/v), more preferably from 90 to 95% (v/v) relative to the total volume of the mixture in the case of ketone, or in an amount comprised from 71 to 99% (v/v), preferably from 80 to 95% (v/v), more preferably from 90% to 95% (v/v) relative to the total volume of the mixture in the case of alcohol.

[0033] The heteropolyacid, or complex acid, as per point a) is preferably phosphotungstic acid, with the formula H.sub.3PW.sub.12O.sub.40, or phosphomolybdic acid, with the formula H.sub.3PMo.sub.12O.sub.40, which are commercially available, or known precursors thereof containing phosphorous and tungsten or phosphorous and molybdenum, such as, for example: sodium tungstate with the formula Na.sub.2WO.sub.4 or sodium molybdate with the formula Na.sub.2MoO.sub.4, both to be mixed with orthophosphoric acid and hydrochloric acid. The lanthanide salt as per point b) preferably has an element selected from ytterbium, europium, terbium and gadolinium as a cation; ytterbium is particularly preferred.

[0034] The corresponding anion is preferably a halide, even more preferably it is chloride. The lanthanide salt can be either in an anhydrous form, in the event that n is equal to 0, or in a hydrated form. In the latter case, said salt preferably contains a number n equal to 6 molecules of water of crystallisation.

[0035] The molar ratio between the component a) and the component b) of the present solution (b/a molar ratio) is preferably comprised in the interval of 0.1-100, preferably 1-50, even more preferably 1-15.

[0036] The concentration of the component b) in the solvent c) is preferably comprised from 0.01 to 250 mM, preferably from 0.1 to 100 mM, even more preferably from 1 to 50 mM. Outside these intervals, in fact, one observes an increase in the nonspecific contrast of the samples, which leads to a reduction in the signal-to-noise ratio.

[0037] In a preferred embodiment of the invention, the solvent c) consists of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is a ketone and wherein, in the case of the water/organic solvent mixture, the organic solvent is present in an amount comprised from 1 to 99% (v/v), preferably from 20 to 99% (v/v), more preferably from 90 to 95% (v/v) relative to the total volume of the water/ketone mixture.

[0038] Said ketone is selected from a ketone with a number of carbon atoms comprised from 1 to 4, preferably from 1 to 3. Preferably, said ketone is acetone.

[0039] In one embodiment of the invention, the solvent c) consists of a water/organic solvent mixture or an organic solvent, wherein said organic solvent is an alcohol and wherein, in the case of the water/organic solvent mixture, the organic solvent is present in an amount comprised from 71 to 99% (v/v), preferably from 80 to 95% (v/v), more preferably from 90 to 95% (v/v) relative to the total volume of the water/alcohol mixture. Said alcohol is selected from an alcohol with a number of carbon atoms comprised from 1 to 4, preferably from 1 to 3. Said alcohol is preferably selected from methanol and ethanol.

[0040] The water and organic solvent mixture has the advantage of optimising the penetration of the contrast agent into the biological sample, and the aqueous part of said mixture makes the preparation of the contrast solution simpler in terms of the dissolution of the above-described components a) and b).

[0041] Optionally, in some embodiments of the invention, in addition to the intrinsic buffering power of the component a), a buffer solution or a strong acid or base can be used to reach the pH interval of 4-6. In the case of a buffer solution, it is selected from the ones known to the person skilled in the art and can be an organic or inorganic buffer, such as, for example, 2-[N-morpholino]ethanesulfonic acid (MES), cacodylate, acetate or formate. In a particularly preferred embodiment, the solution according to the present invention comprises: [0042] a) phosphotungstic acid, with the formula H.sub.3PW.sub.12O.sub.40; [0043] b) ytterbium chloride hexahydrate, with the formula YbCl.sub.3*6H.sub.2O; [0044] c) a solvent consisting of a water/acetone mixture containing from 90% to 95% (v/v) acetone relative to the total volume of the water/acetone mixture;

[0045] The solution described thus far, comprising the components a) to c), has application as a contrast medium for the characterisation of biological samples in a suspension (negative contrast) or embedded in resin, processed for correlative microscopy (CLEM) or conventional electron microscopy (EM).

[0046] In a second aspect, the present invention relates to the use of a compound obtainable from the reaction between the above-described components a) and b), under ambient pressure and temperature conditions and at a pH comprised in the interval of 4-6, as a contrast medium for biological samples for analysis by either correlative microscopy (CLEM) or conventional electron microscopy (EM).

[0047] A compound obtainable from a reaction between the components a) and b) means a chemical species containing atoms of oxygen, phosphorous and M originating from the component a) and Ln originating from the component b), and which manifests itself as thermodynamically stable in the solution of the solvent c) under the aforesaid conditions and can be used both in the correlative microscopy (CLEM) technique and in conventional electron microscopy (EM), preferably in the correlative microscopy (CLEM) technique.

[0048] According to a preferred embodiment, said compound is obtained from the reaction between equimolar amounts of phosphotungstic acid as component a) and ytterbium chloride as component b) and has the formula [YbPW.sub.9O.sub.34].sup.6−. This species is defined as a lanthanide polyoxometalate.

[0049] In a third aspect, the present invention relates to a method for preparing biological samples for analysis by either correlative microscopy (CLEM) or conventional electron microscopy (EM) and comprising the following steps: [0050] i) performing a physical fixation of the biological sample to be analysed, following the high pressure freezing (HPF) method; [0051] ii) performing the technique of substituting water with an organic solvent at cryogenic temperatures (FS—freeze substitution) in a frozen biological sample obtained in step (i), where the substitution medium is the solution comprising the components a) to c) as defined above.

[0052] The solution as per step ii) proves to be particularly effective when it has a concentration of the component b) comprised from 0.01 to 250 mM, preferably from 0.1 to 100 mM, even more preferably from 1 to 50 mM. Outside these intervals, in fact, one observes an increase in the nonspecific contrast of the samples, which leads to a reduction in the signal-to-noise ratio.

[0053] In one embodiment, the solution as per step ii), comprising the components a) to c) according to the present invention, can be obtained starting from a solution comprising the components a) and b) dissolved in a solvent c′), wherein said solvent c′) is an aqueous or hydroalcoholic solvent containing up to 70% (v/v) alcohol, preferably a hydroalcoholic solvent containing up to 70% (v/v) ethanol. Said solution comprising the components a), b) and c′) represents an intermediate solution and is called, for the purposes of the present invention, a “stock solution”. In a preferred embodiment of the method for preparing biological samples according to the present invention, said stock solution is in fact dried, preferably by freeze-drying, and subsequently reconstituted in a suitable amount of the solvent c) as previously defined, thus obtaining the solution comprising the components a)-c) according to the present invention.

EXAMPLES

Example 1

[0054] Preparation of the PTA-YbCl.sub.3 Solution (Stock Solution)

[0055] A solution of phosphotungstic acid (concentration 3.2 mM) in 10 ml of water/ethanol containing 20% (v/v) ethanol was prepared. The pH of this solution was brought to around 5 with sodium hydroxide 1 M. This solution had added to it an equal volume of an ytterbium chloride hexahydrate 48 mM (final concentration) solution in ethanol/water containing 20% (v/v) ethanol. The pH was again adjusted to 5 with sodium hydroxide 1 M. The mixture was kept under stirring overnight at room temperature. The solution thus obtained was called “X solution 1.5” and manifested a precipitate. The precipitate was removed by filtration and the pH was again adjusted to around 5 by adding a 20 mM solution of MES. The solution was then again kept under stirring overnight and again filtered, this time through a membrane with a pore size of 200 nm so as to obtain a solution free of precipitate and called “stock solution”. The stock solution was then stored in the dark at a temperature of 4° C. until the time of use.

Example 2

[0056] Use of the Solution According to the Present Invention as a Contrast Agent for Characterisation by Correlative Microscopy (CLEM) Using the HPF/FS Protocol for Biological Samples.

[0057] The stock solution obtained as per example 1 (0.2 ml) was freeze-dried and reconstituted in 2 ml (“X Sol 1/10”), 3.2 ml (“X Sol 1/16”) or 6 ml (“X Sol 1/30”) of a water/acetone mixture containing 95% (v/v) acetone in order to obtain the solution according to the present invention, in three different concentrations, respectively.

[0058] Each solution was centrifuged and the supernatant was used as the substitution medium in the standard HPF/FS protocol (illustrated in FIG. 1) on line cell stably transfected with a fluorescent protein conjugated to the endoplasmic reticulum. These were used as the standard sample for the correlative protocols. The cells embedded with this protocol were processed by means of the technique of high-pressure freezing/substitution of water with an organic solvent at cryogenic temperatures (HPF/FS). The correlative analysis was performed on sample sections (cut by using an ultramicrotome) and collected on supports for electron microscopy (copper grids). The same grid on which the above-mentioned sections were collected was analysed both by confocal optical microscopy for the selection of the fields of interest and, subsequently, by means of an ultrastructural approach. The correlation between the two types of images originating from the same sample is the basic principle of correlative methods, that is, the association of functional data (marking of the endoplasmic reticulum) with structural data (as the same cells appear positive for the fluorescence signal).

[0059] The various samples obtained were observed using both the confocal optical microscopy technique (FIG. 2) and the transmission electron microscopy TEM (FIG. 3) technique and the results were compared with those obtained using uranyl acetate (“UA”) as the contrast agent or in the absence of a contrast agent (“unstained”).

[0060] FIG. 4 shows the TEM images of the samples with the preferred contrast solution according to the present invention, i.e. the solution obtained starting from the stock solution described in example 1, freeze-dried and reconstituted in acetone in a 1/10 (v/v) ratio relative to the volume of the solution prior to freeze-drying. In these images it is possible to observe how the cellular ultrastructure is extremely well resolved thanks precisely to the increase in contrast efficiency. In particular, in this case, using the solution according to the present invention makes it possible to visualise subcellular details (such as, for example, the cytoskeleton) that are not generally observable using traditional contrast agents, for example based on uranyl acetate.

Example 3

[0061] Use of the Solution According to the Present Invention as a Contrast Agent for Characterisation by Correlative Microscopy (CLEM) with the HPF/FS Protocol for Biological Samples (Drosophila Oocyte)

[0062] An experiment similar to the one described in Example 2 was carried out using Drosophila oocytes as the biological sample and using ethanol as an organic solvent in the place of acetone. The images obtained are shown in FIG. 5.

[0063] The images obtained thus show that the solution of the present invention enables comparable results to be obtained in terms of fluorescence emission, while at the same time ensuring, however, better contrast than when a solution containing uranyl acetate is used, thus proving more effective in the case of characterisation by correlative microscopy (CLEM).