Embedding media for biological samples for imaging by optical and / or electron microscopy

Abstract

The present invention relates to the field of microscopy, preferably the field of electron microscopy. In particular, the present invention relates to an embedding agent for imaging a biological sample by microscopy, comprising: 60% to 99% by weight of a diol dimethacrylate selected from alkylene glycol dimethacrylate and / or oligo(alkylene glycol) dimethacrylate; 0% to 38% by weight of a polyalkylene glycol diacrylate or polyalkylene glycol methacrylate; the polyalkylene glycol diacrylate or polyalkylene glycol methacrylate is optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; at least one additive, preferably comprising at least one heavy metal salt or lanthanide salt; and 0.1% to 2% by weight of a free radical polymerization initiator. The present invention also relates to a conductive material obtained by polymerizing the embedding agent of the present invention, and a preparation method and a kit thereof; the material embedding at least one biological sample. The present invention also relates to a method for imaging a biological sample by microscopy, the method comprising using the embedding agent and / or conductive material of the present invention.

Description

Technical Field

[0001] The present invention relates to the field of microscopy, preferably to the field of optical microscopy and / or electron microscopy. In particular, the present invention relates to an embedding agent for imaging biological samples by microscopy. The embedding agent of the present invention relates to a composition comprising or consisting of a mixture of (i) diol dimethacrylate, (ii) polyalkylene glycol diacrylate and / or polyalkylene glycol methacrylate and (iii) an initiator. The present invention also relates to a conductive material and / or a biological preservation material obtained by polymerizing the embedding agent of the present invention, and a preparation method and a kit thereof; said material embedding at least one biological sample.

[0002] The present invention also relates to a method for imaging a biological sample by microscopy, which method comprises using the embedding agent and / or the conductive material of the present invention. Background Art

[0003] Microscopy refers to a group of techniques used to observe objects and target areas that are inaccessible to the naked eye. Among the well-known microscopy techniques, electron microscopy is useful for observing biological materials. This technique can produce amplitude or phase contrast images due to diffraction, reflection or refraction caused by the direct interaction of electron beams or electromagnetic radiation with the sample to be observed.

[0004] Until now, it has been possible to achieve good amplitude contrast only by adding contrast media which consist essentially of heavy metal salts and fixatives (eg aldehydes).

[0005] High-resolution electron microscopy visualization of any sample also includes complex preparation steps such as fixation, which allows the sample to closely resemble a native sample during microscopic analysis while remaining protected from the high vacuum of electron microscopy.

[0006] Fixation of biological materials can be achieved by chemical methods (addition of fixatives such as aldehydes) or physical methods (ultrafast freezing methods to achieve vitrification or amorphous fixation of water in biological materials).

[0007] In particular, to enable observation of native biological materials after fixation, polymer resins have been developed to replace the water of fixed biological specimens to protect them from the effects of vacuum while strengthening them to allow thin sectioning by ultramicrotomy.

[0008] However, the use of fixatives and contrast agents can lead to: 1) problems with post-treatment of contaminated waste during sample preparation; 2) changes in biological properties; and 3) poor charge dissipation during imaging. In fact, fixatives and heavy metal salts are toxic, mutagenic, carcinogenic and / or radioactive. They partially or completely change the biological properties of fluorescence emission after stimulation or protein activity or mask antigens used for immunoassays. In addition, heavy metal salts only capture electrons during imaging, and do not allow the release of charges accumulated on the sample surface during the process of realizing microscopy film. As a result, charges accumulate locally on the sample surface, resulting in the formation of halos around the sample and hindering complete imaging. Therefore, it is difficult to make film.

[0009] For example, in transmission electron microscopy (TEM) or electron tomography, this overload significantly changes the focal plane and the image is obtained at the wrong focus. In scanning electron microscopy (SEM), electron overload produces a white halo on the sample surface.

[0010] Alternative solutions have been developed. One solution is to reduce the resulting charge effects by using heavy metal salts in large quantities and by improving low electron imaging parameters (Deerinck et al., 2010). However, this method is very harsh, not suitable for many biological samples, and requires working at room temperature, which prevents the optimal ultrastructural preservation achieved by cryofixation. In particular, this method can highlight large parts of the membrane or cell wall, but results in a large extraction that does not include all information related to the rest of the biological components.

[0011] Another solution is to combine the use of heavy metal salts with thin carbon films or metal deposition by evaporation, the latter making it possible to provide an electronic discharge. However, this method requires additional steps to prepare the sample and to evacuate the charge only in the vicinity of the sample. In addition, the amount of heavy metal salts still leads to the need for subsequent waste reprocessing.

[0012] Therefore, there is a need to provide a more efficient method for preparing biological samples for electron microscopy imaging. In particular, there is a need to provide more efficient embedding agents for biological materials that can reduce the need for contrast agents and / or their biological effects while maximizing the contrast of the sample during imaging by electron microscopy to obtain the best quality images possible.

[0013] Among the commercially available resins, The resins have the advantage of hardly sublimating under the action of an electron beam. In addition, the precursor solutions of these resins (i.e. before polymerization) remain fluid at temperatures as low as -35°C or -70°C, depending on the product, making them compatible with rapid freezing and freeze substitution methods for the analysis of vitrified biological samples. To date, rapid freezing is considered the fixation method that is closest to natural biological materials and is widely used for qualitative and quantitative analysis.

[0014] However, The resin is not electrically conductive, which results in unsatisfactory visualization of the sample during electron microscopy analysis, particularly if too little contrast agent is used during sample preparation.

[0015] Therefore, it is also necessary to provide a biomaterial embedding agent that can effectively drain the electric charge accumulated on the sample and / or embedding agent surface during the electron microscopy imaging after embedding at room temperature or low temperature. It is also necessary to provide a more effective biomaterial embedding agent that can keep good stability and / or physical integrity during its electron microscopy imaging. It is also necessary to provide an embedding agent that can significantly reduce the amount of used heavy metal salts or lanthanide salts. Therefore, it is also necessary to provide a biomaterial embedding agent that can retain the best ultrastructure during microscopic analysis.

[0016] Fluorescence microscopy can effectively target biological samples, further optimizing the use of electron microscopy in biological research. The use of fixatives and / or contrast agents significantly reduces the fluorescence and native properties of proteins embedded in the resin. Epoxy resins, for example, interact with the amine groups of proteins, preventing them from further acting as proteins. The resin or acrylate resin does not cross-react with biological materials and preserves its resonant properties.

[0017] Therefore, there is a need to provide a non-biologically interactive embedding agent that can effectively preserve the natural fluorescence of the embedded biological sample while retaining the contrast of the underlying electron microscopy (EM) technique.

[0018] Surprisingly, the Applicant has demonstrated that a hydrophilic resin obtained by polymerization of a monomer mixture selected from glycol dimethacrylates, polyalkylene glycol diacrylates and / or polyalkylene glycol methacrylates provides an effective embedding agent solving the above-mentioned problems of the prior art.

[0019] Advantageously, the ultrastructure and fluorescence of the biological sample included in the embedding medium of the invention are preserved. Advantageously, the imaging contrast obtained with the embedding medium of the invention is greater than that obtained using commercial resins. Summary of the invention

[0020] Therefore, the present invention relates to a conductive embedding agent for a biological sample, comprising:

[0021] - 60 to 99% by weight of a glycol dimethacrylate selected from alkylene glycol dimethacrylates and / or oligo(alkylene glycol) dimethacrylates;

[0022] - polyalkylene glycol diacrylate or polyalkylene glycol methacrylate; the polyalkylene glycol diacrylate or polyalkylene glycol methacrylate is optionally substituted by at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group;

[0023] - at least one additive, preferably comprising at least one heavy metal salt or lanthanide salt; and

[0024] - Free radical polymerization initiators.

[0025] According to one embodiment, a conductive embedding agent for a biological sample comprises:

[0026] - 60 to 99% by weight of a glycol dimethacrylate selected from alkylene glycol dimethacrylates and / or oligo(alkylene glycol) dimethacrylates;

[0027] - 0 to 38% by weight of polyalkylene glycol diacrylate or polyalkylene glycol methacrylate; the polyalkylene glycol diacrylate or polyalkylene glycol methacrylate is optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; and

[0028] - 0.1 to 2% by weight of a free-radical polymerization initiator.

[0029] According to one embodiment, the diol dimethacrylate is selected from ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, propylene glycol dimethacrylate and butylene glycol dimethacrylate; preferably, the diol dimethacrylate is triethylene glycol dimethacrylate.

[0030] According to one embodiment, the polyalkylene glycol diacrylate is selected from polyethylene glycol diacrylate, polypropylene glycol diacrylate and polybutylene glycol diacrylate; preferably, the polyalkylene glycol diacrylate is polypropylene glycol diacrylate.

[0031] According to one embodiment, the polyalkylene glycol methacrylate is selected from polyethylene glycol methacrylate, polypropylene glycol methacrylate and polybutylene glycol methacrylate; the polyalkylene glycol methacrylate is optionally substituted by at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; preferably, the polyalkylene glycol methacrylate is a hydroxypolyethylene glycol methacrylate.

[0032] According to one embodiment, the free radical polymerization initiator is a photoinitiator or a thermal initiator, preferably the free radical polymerization initiator is a photoinitiator, more preferably benzoin methyl ether or 2-hydroxy-2-methyl-1-phenyl-propane-1-one.

[0033] According to one embodiment, the conductive embedding agent comprises:

[0034] - 60 to 99% by weight of triethylene glycol dimethacrylate;

[0035] - from greater than 0% to 38% by weight of polypropylene glycol diacrylate; and

[0036] - 0.1 to 2% by weight of a free-radical polymerization initiator.

[0037] According to one embodiment, the conductive embedding agent comprises:

[0038] - 60 to 99% by weight of triethylene glycol dimethacrylate;

[0039] - from greater than 0% to 38% by weight of hydroxypolyethylene glycol methacrylate; and

[0040] - 0.1 to 2% by weight of a free-radical polymerization initiator.

[0041] According to one embodiment, the conductive embedding agent comprises at least one heavy metal salt, more preferably osmium acetate and / or uranyl acetate.

[0042] According to one embodiment, the conductive embedding medium further comprises at least one biological sample, preferably a vitrified and / or chemically fixed and / or dehydrated biological sample.

[0043] The present invention also relates to a conductive imaging material comprising at least one biological sample embedded in a polymer matrix produced by polymerization of the embedding agent as described above.

[0044] According to one embodiment, the conductive material retains the intrinsic fluorescence, protein activity and / or antigen specificity of the embedded biological sample.

[0045] The present invention also relates to a method for imaging a biological sample by microscopy, comprising the following steps:

[0046] (i-1) fixing and / or dehydrating biological samples;

[0047] (ii) infiltrating the conductive embedding agent defined above into the biological sample of step (i-1);

[0048] (iii) polymerizing the mixture obtained in step (ii) to obtain a conductive imaging material;

[0049] (iv) imaging the conductive imaging material of step (iii) by optical microscopy and / or electron microscopy.

[0050] According to one embodiment, the method further comprises the step of performing sectioning or ultra-thin sectioning on the conductive imaging material.

[0051] The invention also relates to the use of an embedding agent as defined above or an electrically conductive imaging material as defined above for imaging a biological sample by microscopy, preferably by optical microscopy and / or electron microscopy.

[0052] The present invention also relates to a kit for preparing a biological sample to be imaged by microscopy, comprising:

[0053] - a first container comprising (a) a diol dimethacrylate selected from alkylene glycol dimethacrylates and / or oligo(alkylene glycol) dimethacrylates; and (b) a polyalkylene glycol diacrylate or a polyalkylene glycol methacrylate, the polyalkylene glycol diacrylate or the polyalkylene glycol methacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group;

[0054] - a second container comprising a free radical polymerization initiator;

[0055] or contain:

[0056] A container comprising (a) a diol dimethacrylate selected from alkylene glycol dimethacrylates and / or oligo(alkylene glycol) dimethacrylates; (b) a polyalkylene glycol diacrylate or a polyalkylene glycol methacrylate, the polyalkylene glycol diacrylate or the polyalkylene glycol methacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; and (c) a free radical polymerization initiator;

[0057] and mixing devices.

[0058] definition

[0059] In the present invention, the following terms have the following meanings:

[0060] - "About": before a number means plus or minus 10% of the stated numerical value.

[0061] - "Acrylate": refers to a vinyl monomer having at least one CH2=CH-COO- group.

[0062] - "Amino" refers to any compound derived from ammonia NH3 by replacing one or more than one hydrogen atom with an organic group. Amino preferably refers to -NH2, -NHR and -NRR', wherein R and R' are preferably alkyl. Thus, "amino" includes monoalkylamino and dialkylamino.

[0063] - "Biological sample" means any biological material collected from an animal or plant or a living human body, such as a cell extract, cell, tissue or organ. Biological samples do not include implants.

[0064] - "Embedding medium" refers to a medium into which a biological specimen is placed so that the resulting material can be imaged using microscopic techniques, either directly or after cutting using a microtome or ultramicrotome. Embedding medium does not refer to objects or metals used as implants.

[0065] - "Diol": refers to any organic compound having at least one diol group and each hydroxyl group thereof being located on a different carbon atom. According to one embodiment, each hydroxyl group of the diol compound is located at one end of the diol compound. According to one embodiment, the diol has at least one vicinal diol, i.e., two hydroxyl groups are attached to adjacent carbon atoms. Diol compounds include alkylene glycols, such as ethylene glycol, propylene glycol or butylene glycol; oligo(alkylene glycols), such as diethylene glycol, triethylene glycol or tetraethylene glycol; and polyalkylene glycols, such as poly(ethylene glycol), poly(propylene glycol) or poly(butylene glycol).

[0066] - "Diol acrylate": refers to any diol compound in which one of the two hydroxyl functional groups of the diol is reacted with acrylic acid or acrylic halide to provide a compound having a hydroxyl functional group and an acrylate group. The term "diol acrylate" includes alkylene glycol acrylates, oligo(alkylene glycol) acrylates and polyalkylene glycol acrylates.

[0067] - "Diol diacrylate": refers to any diol compound wherein the two hydroxyl functional groups of the diol are reacted with acrylic acid or acrylic halide to provide a compound having two acrylate groups. The term "diol diacrylate" includes alkylene glycol diacrylates, oligo(alkylene glycol) diacrylates and polyalkylene glycol diacrylates.

[0068] - "Diol dimethacrylate": refers to any diol compound wherein the two hydroxyl functional groups of the diol are reacted with methacrylic acid or methacrylic acid halide to provide a compound having two methacrylate groups. The term "diol dimethacrylate" includes alkylene glycol dimethacrylates, oligo(alkylene glycol) dimethacrylates and polyalkylene glycol dimethacrylates.

[0069] - "Diol methacrylate": refers to any diol compound wherein one of the two hydroxyl functional groups of the diol is reacted with methacrylic acid or methacrylic acid halide to provide a compound having a hydroxyl functional group and a methacrylate group. The term "diol methacrylate" includes alkylene glycol methacrylates, oligo(alkylene glycol) methacrylates and polyalkylene glycol methacrylates.

[0070] - "Heavy metal salt": refers to any salt comprising a heavy metal (i.e. a metal with a relatively high density, atomic weight or atomic number). According to one embodiment, the heavy metal is selected from iron, cobalt, zinc, ruthenium, silver, indium, cadmium, mercury, lead, uranium, osmium and copper.

[0071] - "Hydrophilic": defines a molecule or part of a molecule that has a generally polarized charge and is capable of hydrogen bonding, making it more soluble in water than in oil or other solvents.

[0072] - "Hydroxyl": refers to the chemical functional group -OH.

[0073] - "Initiator": refers to any compound capable of initiating a polymerization reaction, preferably a free radical polymerization. In the present invention, the initiator provides a free radical entity capable of reacting with the first vinyl monomer unit, thereby initiating the growth of the polymer chain.

[0074] - refers to commercially available embedding media based on acrylate and / or methacrylate resins. HM 20 refers to a non-polar embedding agent comprising or consisting of 2-methyl-1,2-ethanediylbis(oxy-2,1-ethanediyl) 2-acrylate (CAS 106-16-0); 2-methylethyl 2-propionate (CAS 97-63-2); n-hexyl methacrylate and optionally 4-methoxyphenol and / or acetophenone-2-methoxy-2-phenyl (CAS 3524-62-7). According to one embodiment, the term "Lowicryl" includes commercially available products HM 20, HM 21 and HM 23.

[0075] - "Macromer" means a polymer chain having at least one terminal group capable of acting as a monomer. According to one embodiment, the term

[0076] - "Matrix": refers to a chemical network, preferably an organic chemical network, more preferably an organic polymer network. According to one embodiment, the network is covalently cross-linked.

[0077] - "Imaging material": refers to a solid or semi-solid material that embeds a biological sample and is suitable for analysis by microscopic techniques.

[0078] - "Methacrylate": refers to a vinyl monomer having at least one CH2=C(CH3)-COO- group.

[0079] - “Microscopy”: refers to the general field covering the use of microscopic techniques to image objects.

[0080] - "Microtomy": refers to a method of preparing an object for microscopic investigation comprising the use of a microtome or ultramicrotome, i.e. a device suitable for thinly slicing a sample to be analyzed by microscopy. According to one embodiment, the term "microtomy" comprises focused ion beam milling techniques.

[0081] - “Electron microscopy”: refers to microscopy techniques that use a beam of accelerated electrons as a source of illumination for the sample and provide a magnified image.

[0082] - "Optical microscopy" refers to microscopy techniques that use a beam of photons of controlled wavelength as a source of illumination of the sample and provide a magnified image.

[0083] - "Electrically conductive": refers to any material or compound capable of conducting electrons. According to one embodiment, the term "electrically conductive" refers to a material or compound capable of evacuating electrons accumulated on its surface.

[0084] - "Freeze substitution" refers to the process of dehydration of biological samples, which is carried out at a sufficiently low temperature to avoid the formation of ice crystals and avoid the destructive effects observed after dehydration of the same samples at ambient temperature. In the present invention, the freeze substitution process refers to the process of first dissolving the frozen water of the sample by an organic solvent, which optionally contains a chemical fixative; then, embedding the sample in a resin; and finally, gradually heating the sample for microscopic imaging.

[0085] - "High-pressure freezing" refers to a cryofixation method in which biological samples are subjected to high pressure, preferably at a pressure of 2000 to 2700 bar, more preferably at a pressure of about 2048 bar.

[0086] - "Carbonyl" refers to the chemical functional group -C(O)-.

[0087] - "Polymer": refers to a macromolecular compound composed of repeating units (monomers). According to one embodiment, the term "polymer" refers to an organic macromolecular compound composed of repeating units.

[0088] - "Photoinitiator": refers to any compound capable of initiating a polymerization reaction, preferably a free radical polymerization, when exposed to radiation (UV or visible light). In the present invention, when exposed to radiation, the photoinitiator provides a free radical entity capable of reacting with the first vinyl monomer unit, thereby initiating the growth of the polymer chain.

[0089] - "Free radical polymerization" refers to any polymerization technique in which the growth of polymer chains is achieved by the continuous addition of free radical monomers. In the present invention, the expression "free radical polymerization" refers to a method for providing a polymer, which comprises the following three steps: (1) initiation, (2) propagation and (3) termination, which is well known to the skilled person.

[0090] - "Thermal initiator": refers to any compound capable of initiating polymerization, preferably free radical polymerization, when exposed to heat. In the present invention, when exposed to heat, the thermal initiator provides a free radical entity capable of reacting with the first vinyl monomer unit, thereby initiating the growth of the polymer chain.

[0091] - "Ultramtomy" means a method of preparing an object for study by microscopy which comprises the use of an ultramicrotome (i.e. a device equipped with a diamond or glass knife suitable for slicing a sample ultrathinly for microscopic analysis).

[0092] - "Vitrification": refers to the process of converting biological materials into a solid containing amorphous water, in which all cellular components are kept in a near-living state. In the present invention, the expression "vitrified biological sample" refers to a biological sample obtained after vitrification. DETAILED DESCRIPTION OF THE INVENTION

[0094] Embedding agent

[0095] The present invention relates to an embedding agent, preferably an embedding agent for biological samples, even more preferably an embedding agent for wetting and impregnating biological samples. According to one embodiment, the present invention relates to an embedding agent for imaging biological samples, preferably by microscopy, more preferably by electron microscopy. According to one embodiment, the embedding agent is electrically conductive.

[0096] According to one embodiment, the embedding agent is a composition comprising or consisting of an organic compound.

[0097] According to one embodiment, the embedding agent comprises or is constituted as a polymer precursor preparation.According to one embodiment, the embedding agent comprises or is constituted as a monomer mixture and optionally an initiator.

[0098] According to one embodiment, the embedding agent comprises or is constituted as a monomer mixture, wherein at least one monomer has the formula (I):

[0099]

[0100] in:

[0101] R1 represents H, a linear or branched alkyl group, preferably R1 represents H or a methyl group;

[0102] R2 represents H, methacryloyl, acryloyl, straight chain or branched alkyl, preferably R1

[0103] represents H, methacryloyl or acryloyl;

[0104] R a , R b , R c and R d Each is independently selected from H, a linear or branched alkyl group, preferably selected from H or a methyl group;

[0105] n is a positive integer.

[0106] According to one embodiment, the embedding agent comprises or is constituted as a monomer mixture, which includes:

[0107] - A monomer having formula (II-A):

[0108]

[0109] - A monomer having formula (II-B):

[0110]

[0111] and / or

[0112] - A monomer having formula (II-C):

[0113]

[0114] in:

[0115] R1 represents H, a linear or branched alkyl group, preferably R1 represents H or a methyl group;

[0116] R2 represents H, methacryloyl, acryloyl, a linear or branched alkyl group, preferably R1 represents H, methacryloyl or acryloyl;

[0117] R a represents H, a linear or branched alkyl group; preferably selected from H or methyl;

[0118] n1 is a positive integer from 1 to 5, preferably 3;

[0119] n2 and n3 each independently represent a positive integer greater than 5, preferably 10 to 100, more preferably 10 to 40.

[0120] According to one embodiment, the monomer mixture comprises or consists of at least one monomer, preferably at least one free radical polymerizable monomer, more preferably at least one hydrophilic free radical polymerizable monomer. According to one embodiment, the embedding agent comprises or consists of at least one hydrophilic monomer. According to one embodiment, the embedding agent comprises or consists of a mixture of two free radical polymerizable monomers.

[0121] According to one embodiment, the free-radically polymerizable monomers are chosen from acrylates, methacrylates, diacrylates and / or dimethacrylates, said groups being optionally substituted by at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group.

[0122] According to one embodiment, the embedding agent comprises:

[0123] - polyalkylene glycol (di)acrylates or polyalkylene glycol (di)methacrylates, the polyalkylene glycol diacrylates or polyalkylene glycol methacrylates being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; and

[0124] -Initiator.

[0125] According to one embodiment, the embedding agent comprises:

[0126] - alkylene glycol dimethacrylate;

[0127] - polyalkylene glycol diacrylate or polyalkylene glycol methacrylate, said polyalkylene glycol diacrylate or polyalkylene glycol methacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; and

[0128] -Initiator.

[0129] According to one embodiment, the embedding agent does not comprise dimethylaminoethyl methacrylate (DEMA), hydroxyethyl methacrylate (HEMA), polymethyl methacrylate (PMMA), poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA) or polyethylene glycol (PEG).

[0130] According to one embodiment, the monomer mixture comprises or consists of one diacrylate and one dimethacrylate, preferably one diol diacrylate and one diol dimethacrylate; the diol diacrylate and / or diol dimethacrylate are optionally substituted by at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group.

[0131] According to one embodiment, the monomer mixture comprises or consists of one or more than one alkylene glycol dimethacrylate and one polyalkylene glycol diacrylate, the alkylene glycol dimethacrylate and / or the polyalkylene glycol diacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group.

[0132] According to one embodiment, the monomer mixture comprises or consists of an alkylene glycol dimethacrylate and a polyalkylene glycol methacrylate, said alkylene glycol dimethacrylate and / or polyalkylene glycol methacrylate being optionally substituted by at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group.

[0133] According to one embodiment, the alkylene glycol dimethacrylate is selected from ethylene glycol dimethacrylate, propylene glycol dimethacrylate and butylene glycol dimethacrylate; preferably, the alkylene glycol dimethacrylate is ethylene glycol dimethacrylate.

[0134] According to one embodiment, the polyalkylene glycol diacrylate is selected from polyethylene glycol diacrylate, polypropylene glycol diacrylate and polybutylene glycol diacrylate; preferably, the polyalkylene glycol diacrylate is polypropylene glycol diacrylate.

[0135] According to one embodiment, the polyalkylene glycol methacrylate is selected from polyethylene glycol methacrylate, polypropylene glycol methacrylate and polybutylene glycol methacrylate, and the polyalkylene glycol methacrylate is optionally substituted by at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; preferably, the polyalkylene glycol methacrylate is a hydroxy polyethylene glycol methacrylate.

[0136] According to one embodiment, the monomer mixture comprises or consists of triethylene glycol dimethacrylate (TGdMA) and polypropylene glycol diacrylate (PPGdA). According to one embodiment, the embedding agent comprises or consists of triethylene glycol dimethacrylate, polypropylene glycol diacrylate and an initiator.

[0137] According to one embodiment, the initiator is a free radical polymerization initiator. According to one embodiment, the initiator is a photoinitiator. According to one embodiment, the initiator is a thermal initiator.

[0138] According to one embodiment, the photoinitiator is selected from 2-tert-butyl anthraquinone, camphorquinone, diphenyl (2,4,6-trimethylbenzoyl) phosphine oxide, 9,10-phenanthrenequinone, phenyl bis (2,4,6-trimethylbenzoyl) phosphine oxide, 2-benzyl-2- (dimethylamino) -4'-morpholino phenyl ketone, 3,6-bis (2-methyl-2-morpholino propionyl) -9-octyl carbazole, 4'-tert-butyl- 2',6'-dimethylacetophenone, 2,2-diethoxyacetophenone, 4'-ethoxyacetophenone, 3'-hydroxyacetophenone, 4'-hydroxyacetophenone, 1-hydroxycyclohexylpropiophenone, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, 2-hydroxy-2-methylpropiophenone, 2-methyl-4'-(methylthio)-2-morpholinopropiophenone, 4'-phenoxyacetophenone, benzoin , benzoin ethyl ether, benzoin methyl ether, 4,4'-dimethylbenzoin, 4,4'-dimethylbenzophenone, benzophenone, benzoylbiphenyl, 4,4'-bis(diethylamino)benzophenone, 4,4'-dihydroxybenzophenone, 3,4-dimethylbenzophenone, 3-hydroxybenzophenone, 4-hydroxybenzophenone, 2-methylbenzophenone, 3-methylbenzophenone, 4-methylbenzophenone, benzene Methyl formylformate, Michell ketone, 1-chloro-4-propoxy-9H-thioxanthen-9-one, 2-chlorothioxanthen-9-one, 2,4-diethyl-9H-thioxanthen-9-one, isopropyl-9H-thioxanthen-9-one, 10-methylphenothiazine, thioxanthen-9-one, diaryliodonium hexafluorophosphate, diaryliodonium hexafluoroantimonate, triaryliodonium hexafluorophosphate, hexafluorophosphate, and triarylsulfonium hexafluoroantimonate.

[0139] According to one embodiment, the thermal initiator is selected from azo compounds such as 2,2'-azobis(isobutyronitrile) (AIBN), 4,4'-azobis-(4-cyanovaleric acid), 1,1'-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropylamidine) dihydrochloride, 2,2'-azobis(2-methylpropionitrile); and organic peroxides such as benzoyl peroxide (BPO).

[0140] According to one embodiment, the alkylene glycol dimethacrylate or any derivative thereof is 60% to 99% by weight, preferably 65% ​​to 99% by weight, 70% to 99% by weight, 75% to 99% by weight, 80% to 99% by weight, 85% to 99% by weight, 90% to 99% by weight. According to one embodiment, the alkylene glycol dimethacrylate is 60% to 99% by weight, preferably 60% to 90% by weight, 60% to 85% by weight, 60% to 80% by weight, 60% to 75% by weight, 60% to 70% by weight, of the total weight of the embedding agent. According to one embodiment, the alkylene glycol dimethacrylate is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% by weight of the total weight of the embedding agent. According to one embodiment, the alkylene glycol dimethacrylate or any derivative thereof is 70% to 90% by weight, 75% to 85% by weight, more preferably 80% by weight of the total weight of the embedding agent.

[0141] According to one embodiment, the alkylene glycol dimethacrylate or any derivative thereof is 0.1% to 100% by weight of the total weight of the embedding agent; preferably 0.1% to 90% by weight; 1% to 90% by weight; 10% to 90% by weight; 15% to 90% by weight; 20% to 90% by weight; 25% to 90% by weight; 30% to 90% by weight; 35% to 90% by weight; 40% to 90%; 45% to 90%; 50% to 90%; 55% to 90%; 60% to 90%; 65% to 90% by weight; 70% to 90% by weight; 75% to 90% by weight; 80% to 90% by weight; or 85% to 90% by weight. According to one embodiment, the alkylene glycol dimethacrylate is 0.1% to 99% by weight of the total weight of the embedding agent; preferably 0.1% to 95% by weight, 0.1% to 90% by weight, 0.1% to 85% by weight, 0.1% to 80% by weight, 0.1% to 75% by weight, 0.1% to 70% by weight, 0.1% to 65% by weight, 0.1% to 60% by weight, 0.1% to 55% by weight, 0.1% to 50% by weight, 0.1% to 45% by weight, 0.1% to 40% by weight, 0.1% to 35% by weight, from 0.1% to 30% by weight, 0.1% to 25% by weight, 0.1% to 20% by weight, 0.1% to 15% by weight, 0.1% to 10% by weight or 0.1% to 5% by weight.

[0142] According to one embodiment, the polyalkylene glycol diacrylate or any derivative thereof is greater than 0% to 38% by weight, preferably 1% to 38% by weight, 5% to 38% by weight, 10% to 38% by weight, 15% to 38% by weight, 20% to 38% by weight, 25% to 38% by weight, 30% to 38% by weight, of the total weight of the embedding agent. According to one embodiment, the polyalkylene glycol diacrylate or any derivative thereof is greater than 0% to 38% by weight, preferably 1% to 35% by weight, 1% to 30% by weight, 1% to 25% by weight, 1% to 20% by weight, 1% to 15% by weight, 1% to 10% by weight, 1% to 5% by weight, of the total weight of the embedding agent.

[0143] According to one embodiment, the polyalkylene glycol diacrylate or any derivative thereof is greater than 0% to 50% by weight, preferably 0.1% to 50% by weight, 5% to 50% by weight, 10% to 50% by weight, 15% to 50% by weight, 20% to 50% by weight, 25% to 50% by weight, 30% to 50% by weight, 35% to 50% by weight, 40% to 50% by weight or 45% to 50% by weight of the total weight of the embedding agent. According to one embodiment, the polyalkylene glycol diacrylate is greater than 0% to 50% by weight, preferably 0.1% to 45% by weight, 0.1% to 40% by weight, 0.1% to 35% by weight, 0.1% to 30% by weight, 0.1% to 25% by weight, 0.1% to 20% by weight, 0.1% to 15% by weight, 0.1% to 10% by weight or 0.1% to 5% by weight of the total weight of the embedding agent. According to one embodiment, the polyalkylene glycol diacrylate is greater than 0% to 50% by weight, preferably 1% to 40% by weight, 5% to 30% by weight, more preferably 10% to 25% by weight, more preferably 20% by weight of the total weight of the embedding agent.

[0144] According to one embodiment, the polyalkylene glycol methacrylate or any derivative thereof is greater than 0% to 38% by weight, preferably 1% to 38% by weight, 5% to 38% by weight, 10% to 38% by weight, 15% to 38% by weight, 20% to 38% by weight, 25% to 38% by weight, 30% to 38% by weight of the total weight of the embedding agent. According to one embodiment, the polyalkylene glycol methacrylate or any derivative thereof is greater than 0% to 38% by weight, preferably 1% to 35% by weight, 1% to 30% by weight, 1% to 25% by weight, 1% to 20% by weight, 1% to 15% by weight, 1% to 10% by weight, 1% to 5% by weight of the total weight of the embedding agent.

[0145] According to one embodiment, the molar weight of the alkylene glycol diacrylate or any derivative thereof is from 100 kDa to 500 kDa; preferably from 100 kDa to 400 kDa, from 100 kDa to 300 kDa, from 100 kDa to 200 kDa. According to one embodiment, the molar weight of the alkylene glycol diacrylate or any derivative thereof is about 150 kDa, preferably 150 kDa. According to one embodiment, the molar weight of triethylene glycol dimethacrylate (TGdMA) is about 150 kDa, preferably 150 kDa.

[0146] According to one embodiment, the molar weight of the polyalkylene glycol diacrylate or any derivative thereof is 500 kDa to 1500 kDa, preferably 500 kDa to 100 kDa, 700 kDa to 900 kDa, 750 kDa to 850 kDa. According to one embodiment, the molar weight of the polyalkylene glycol diacrylate or any derivative thereof is about 800 kDa. According to one embodiment, the molar weight of the polyalkylene glycol diacrylate or any derivative thereof is 800 kDa. According to one embodiment, the molar weight of the polypropylene glycol diacrylate is about 800 kDa, preferably 800 kDa.

[0147] According to one embodiment, the embedding agent comprises or consists of:

[0148] - 79% by weight of an alkylene glycol dimethacrylate, preferably triethylene glycol dimethacrylate;

[0149] - 20% by weight of a polyalkylene glycol diacrylate, preferably polypropylene glycol diacrylate; and

[0150] - 1% by weight of a free radical polymerization initiator, preferably a free radical polymerization photoinitiator, more preferably benzoin methyl ether.

[0151] According to one embodiment, the embedding agent comprises:

[0152] - 60 to 99% by weight of triethylene glycol dimethacrylate;

[0153] - from greater than 0% to 38% by weight of polypropylene glycol methacrylate; and

[0154] - 0.1 to 2% by weight of a free-radical polymerization initiator; preferably benzoin methyl ether.

[0155] According to one embodiment, the embedding agent comprises:

[0156] - 98 to 99.9% by weight of triethylene glycol dimethacrylate;

[0157] - 0.1 to 2% by weight of a free-radical polymerization initiator; preferably benzoin methyl ether.

[0158] According to one embodiment, the embedding agent comprises:

[0159] - 60 to 99% by weight of triethylene glycol dimethacrylate;

[0160] - from greater than 0% to 38% by weight of hydroxypolypropylene glycol methacrylate; and

[0161] - 0.1 to 2% by weight of a free-radical polymerization initiator; preferably benzoin methyl ether.

[0162] According to one embodiment, the embedding medium further comprises a solvent, preferably a volatile solvent, more preferably an alcohol, such as ethanol. According to one embodiment, the embedding medium further comprises acetone.

[0163] According to one embodiment, the embedding agent further comprises at least one additive selected from contrast agents and / or compounds for freeze substitution, such as fixatives and / or solvents, preferably comprising at least one heavy metal salt, more preferably comprising silver nitrate, osmium acetate and / or uranyl acetate. According to one embodiment, the embedding agent further comprises at least one additive, which accounts for greater than 0% to 5% by weight of the total weight of the embedding agent. According to one embodiment, the embedding agent comprises 0.5% by weight of the total weight of the embedding agent. According to one embodiment, the embedding agent comprises 0.01% by weight of the total weight of the embedding agent. According to one embodiment, the compound for freeze substitution is selected from acetone, anhydrous acetone, uranyl acetate, tannic acid, aldehydes such as glutaraldehyde or paraformaldehyde; and any combination thereof. According to one embodiment, the fixative is selected from osmium, osmium tetroxide, uranyl acetate, glutaraldehyde, paraformaldehyde or any combination thereof. According to one embodiment, the contrast agent is selected from uranyl acetate, lead derivatives, silver derivatives, osmium derivatives, lanthanide salts, polyphenols contained in tannins, gallic acid and tannic acid.

[0164] According to one embodiment, the embedding medium further comprises a hydrophilic compound.

[0165] According to one embodiment, the embedding medium further comprises at least one biological sample, preferably a vitrified biological sample and / or a chemically fixed biological sample and / or a dehydrated biological sample. According to one embodiment, the biological sample is a cell extract. According to one embodiment, the biological sample is a cell. According to one embodiment, the biological sample is a tissue. According to one embodiment, the biological sample is an organ. According to one embodiment, the biological sample is a HeLa cell. According to one embodiment, the biological sample is a melanocyte.

[0166] According to one embodiment, the embedding agent is a liquid. According to one embodiment, the embedding agent is a semisolid. According to one embodiment, the embedding agent is polymerizable at low temperatures, preferably at a temperature below 0°C, preferably at a temperature of -90°C to -10°C, more preferably at a temperature of -40°C to -20°C. According to one embodiment, the embedding agent is polymerizable at a temperature of -70°C to -20°C, preferably at a temperature of -35°C, -40°C or -45°C. According to one embodiment, the embedding agent is polymerizable at a temperature of -90°C to -10°C, preferably -80°C to -10°C, -70°C to -10°C, -60°C to -10°C, -50°C to -10°C, -40°C to -10°C, -30°C to -10°C, or -20°C to -10°C. According to one embodiment, the embedding agent is polymerizable at a temperature of -90°C to -10°C, preferably -90°C to -20°C, -90°C to -30°C, -90°C to -40°C, -90°C to -50°C, -90°C to -60°C, -90°C to -70°C or -90°C to -80°C.

[0167] According to one embodiment, the embedding medium is not or does not comprise Resin. According to one embodiment, the embedding agent does not contain phenol or its derivatives. According to one embodiment, the inorganic material does not contain methoxyphenol. According to one embodiment, the inorganic material does not contain alkyl methacrylate. According to one embodiment, the inorganic material does not contain hexyl methacrylate. According to one embodiment, the embedding agent does not contain bisphenol A or its derivatives. According to one embodiment, the inorganic material does not contain ethoxylated bisphenol A dimethacrylate.

[0168] Imaging Materials

[0169] The present invention also relates to a support material for microscopy. According to one embodiment, the support material is a material for microscopy, preferably an embedding material for a sample to be imaged (referred to as "imaging material"). According to one embodiment, the imaging material of the present invention is produced by polymerization of an embedding agent as described above.

[0170] According to one embodiment, the imaging material of the present invention is electrically conductive. According to one embodiment, the imaging material does not accumulate any electrons on its surface during microscopic analysis. According to one embodiment, the imaging material of the present invention is antistatic.

[0171] According to one embodiment, the imaging material of the present invention is transparent. According to one embodiment, the imaging material of the present invention is semi-solid or solid. According to one embodiment, the imaging material of the present invention comprises or is constituted as a cross-linked polymer matrix and optionally at least one biological sample. According to one embodiment, the imaging material of the present invention retains the physical properties of the embedded biological sample, such as its fluorescence and / or retains its protein ultrastructure. According to one embodiment, the imaging material of the present invention retains the protein ultrastructure of the embedded biological sample, so that the detection of antibodies used for immunodetection can be imaged by microscopic techniques.

[0172] According to one embodiment, the imaging material comprises at least one biological sample embedded in a polymer matrix resulting from polymerization of an embedding agent as described above.

[0173] According to one embodiment, the imaging material does not comprise epoxy resin. According to one embodiment, the imaging material does not comprise epoxy groups. According to one embodiment, the imaging material does not comprise poly(arylene ether).

[0174] Method for producing embedding agent

[0175] The present invention also relates to a method for producing the embedding agent described above.

[0176] According to one embodiment, the method comprises or consists of mixing:

[0177] - at least one monomer as defined above, preferably at least one free radical polymerizable monomer, more preferably at least one hydrophilic free radical polymerizable monomer; and

[0178] - an initiator as described above.

[0179] According to one embodiment, the method comprises or consists of mixing:

[0180] - two monomers as defined above, preferably two free radical polymerizable monomers, more preferably two hydrophilic free radical polymerizable monomers to obtain a monomer mixture; and

[0181] -Optionally an initiator as described above.

[0182] According to one embodiment, the method comprises or consists of mixing:

[0183] - alkylene glycol dimethacrylate;

[0184] - polyalkylene glycol diacrylate or polyalkylene glycol methacrylate, said polyalkylene glycol diacrylate or polyalkylene glycol methacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; and

[0185] - an initiator as described above.

[0186] According to one embodiment, the method further comprises adding a biological sample, such as a cell extract, a cell, a tissue and / or an organ; preferably adding a vitrified biological sample and / or a dehydrated biological sample. According to one embodiment, the method further comprises adding an additive, such as a heavy metal salt. According to one embodiment, the method further comprises adding silver nitrate and / or uranyl acetate.

[0187] According to one embodiment, the method for preparing the embedding agent is carried out at a temperature of 0°C to 40°C; preferably at a temperature of 5°C to 30°C, 10°C to 30°C, 15°C to 30°C, 20°C to 30°C, 25°C to 30°C; more preferably, the method for preparing the embedding agent is carried out at a temperature of about 25°C.

[0188] Method for producing imaging material

[0189] The present invention also relates to a method for producing an imaging material as described above.

[0190] According to one embodiment, the method comprises:

[0191] (a) providing an embedding medium as defined above; and

[0192] (c) Polymeric embedding agent.

[0193] According to one embodiment, the method further comprises infiltrating an embedding agent on the biological sample. According to one embodiment, the method comprises:

[0194] (a) providing an embedding medium as defined above;

[0195] (b) impregnating an embedding agent onto the biological sample to provide an embedded biological sample; and

[0196] (c) polymerizing the embedded biological sample of step (b).

[0197] According to one embodiment, the method further comprises vitrifying the biological sample. According to one embodiment, the method comprises:

[0198] (a-0) vitrifying the biological sample to obtain a vitrified biological sample;

[0199] (a) providing an embedding medium as defined above;

[0200] (b) infiltrating an embedding agent onto the vitrified biological sample of step (a-0) to provide an embedded biological sample; and

[0201] (c) polymerizing the embedded biological sample of step (b).

[0202] According to one embodiment, the method further comprises fixing and dehydrating the vitrified biological sample of step (a-0). According to one embodiment, the method comprises:

[0203] (a-0) vitrifying the biological sample to obtain a vitrified biological sample;

[0204] (a-1) fixing and dehydrating the vitrified biological sample of step (a-0);

[0205] (a) providing an embedding medium as defined above;

[0206] (b) infiltrating an embedding agent on the biological sample of step (a-1) to provide an embedded biological sample; and

[0207] (c) polymerizing the embedded biological sample of step (b).

[0208] According to another embodiment, the method further comprises fixing and dehydrating the biological sample. According to one embodiment, the method comprises:

[0209] (a-1) fixing and dehydrating the biological sample;

[0210] (a) providing an embedding medium as defined above;

[0211] (b) infiltrating an embedding agent on the biological sample of step (a-1) to provide an embedded biological sample; and

[0212] (c) polymerizing the embedded biological sample of step (b).

[0213] According to one embodiment, step (a-0) is performed by a low temperature method known to those skilled in the art, preferably by a high pressure freezing method.

[0214] According to one embodiment, step (a-1) is carried out by any method known to those skilled in the art, preferably by freeze substitution. According to one embodiment, step (a-1) is carried out by using at least one fixative, preferably selected from osmium, osmium tetroxide, uranyl acetate, paraformaldehyde and aldehydes such as glutaraldehyde, or any combination thereof. According to one embodiment, step (a-1) is carried out by using at least one contrast agent, preferably selected from heavy metal salts. According to one embodiment, step (a-1) is carried out by using at least one contrast agent, preferably selected from lanthanide salts. According to one embodiment, the contrast agent is selected from uranyl acetate, lead derivatives, silver derivatives, osmium derivatives, lanthanide salts, polyphenols contained in tannins, gallic acid and tannic acid. According to one embodiment, step (a-1) is carried out at a temperature of -90°C to -30°C. According to one embodiment, step (a-1) is carried out at a temperature of -70°C to -20°C, preferably at a temperature of -35°C, -40°C or -45°C. According to one embodiment, step (a-1) is carried out at a temperature of -90°C to -30°C, preferably -80°C to -40°C, or -60°C to -50°C.

[0215] According to one embodiment, step (a) is performed according to the above-mentioned method for preparing an embedding agent.

[0216] According to one embodiment, the polymerization is carried out at a temperature of -90°C to -10°C, preferably at a temperature of -40°C to -20°C. According to one embodiment, the polymerization is carried out at a temperature of -70°C to -20°C, preferably at a temperature of -35°C, -40°C or -45°C. According to one embodiment, the polymerization is carried out at a temperature of -90°C to -10°C, preferably -80°C to -10°C, -70°C to -10°C, -60°C to -10°C, -50°C to -10°C, -40°C to -10°C, -30°C to -10°C or -20°C to -10°C. According to one embodiment, the polymerization is carried out at a temperature of -90°C to -10°C, preferably -90°C to -20°C, -90°C to -30°C, -90°C to -40°C, -90°C to -50°C, -90°C to -60°C, -90°C to -70°C or -90°C to -80°C.

[0217] According to one embodiment, the polymerization is initiated by heating and / or by irradiation, preferably by UV or visible light irradiation.

[0218] According to one embodiment, the weight of the contrast agent is 0.005 wt % to 0.1 wt %, preferably 0.005 wt % to 0.05 wt %, 0.005 wt % to 0.015 wt % of the total weight of the imaging material. According to one embodiment, the contrast agent is about 0.01 wt % of the total weight of the imaging material. According to one embodiment, the contrast agent is 0.01 wt % of the total weight of the imaging material.

[0219] Methods for imaging biological samples by (electron) microscopy

[0220] The invention also relates to a method for imaging a biological sample by microscopy, preferably by optical and / or electron microscopy.

[0221] According to one embodiment, the method of the present invention comprises or consists of the following steps:

[0222] - providing an imaging material as defined above, preferably by a process for the manufacture of an imaging material as defined above; and

[0223] - imaging the imaging material, preferably by microscopy, more preferably by optical microscopy and / or electron microscopy.

[0224] According to one embodiment, the method of the present invention comprises or consists of the following steps:

[0225] (i-0) vitrifying the biological sample, preferably by high pressure freezing, rapid freezing, jet freezing and / or impact freezing;

[0226] (ii) infiltrating the vitrified biological sample of step (i-0) with an embedding medium as defined above, preferably by using a freeze substitution protocol;

[0227] (iii) polymerizing the mixture obtained in step (ii) to obtain a conductive imaging material;

[0228] (iv) imaging the conductive imaging material of step (iii) by optical microscopy and / or electron microscopy.

[0229] According to one embodiment, the method of the present invention comprises or consists of the following steps:

[0230] (ii) infiltrating an embedding medium as defined above onto the biological sample, preferably by using a freeze substitution protocol;

[0231] (iii) polymerizing the mixture obtained in step (ii) to obtain a conductive imaging material;

[0232] (iv) imaging the conductive imaging material of step (iii) by optical microscopy and / or electron microscopy.

[0233] According to one embodiment, the method further comprises the step of performing sectioning or ultra-thin sectioning on the conductive imaging material.

[0234] According to one embodiment, the method further comprises a step of fixing and / or dehydrating the biological sample (step i-1), preferably after step (i-0). According to one embodiment, the method further comprises a step of fixing and / or dehydrating the vitrified biological sample (step i-1), preferably after step (i-0). According to one embodiment, step (i-1) comprises the use of a solvent, preferably acetone and / or ethanol; the solvent comprises a fixative and / or a contrast agent. According to one embodiment, step (i-1) is carried out at a temperature of -90°C to -30°C.

[0235] According to one embodiment, step (iv) is implemented by microscopy; preferably by light microscopy, optical microscopy, fluorescence microscopy and / or electron microscopy; more preferably by electron microscopy. According to one embodiment, step (iv) is implemented by correlative microscopy. According to one embodiment, step (iv) is implemented by scanning electron microscopy (SEM). According to one embodiment, step (iv) is implemented by transmission electron microscopy (TEM). According to one embodiment, step (iv) is implemented by correlative optical and electron microscopy (CLEM). According to one embodiment, step (iv) is implemented by epifluorescence, confocal, super-resolution or structured illumination microscopy (SIM).

[0236] use

[0237] The invention also relates to the use of the embedding agent or imaging material as defined above in microscopy, preferably in light microscopy, fluorescence microscopy and / or electron microscopy; more preferably in electron microscopy. According to one embodiment, the embedding agent or imaging material as defined above is useful in the relevant microscopy technique.

[0238] According to one embodiment, the embedding agent and / or imaging material as defined above is useful for imaging biological samples, such as cell extracts, cells, tissues and / or organs; preferably for imaging vitrified biological samples.

[0239] According to one embodiment, the embedding agent and / or imaging material as defined above is useful for specifically targeting cell structures and / or melanin. According to one embodiment, the embedding agent and / or imaging material as defined above comprising a silver salt such as silver nitrate is useful for specifically targeting cell structures, preferably specifically targeting cell structures and / or melanin labeled using gold-coupled antibodies. According to one embodiment, the embedding agent and / or imaging material as defined above comprising a silver salt such as silver nitrate is useful for specifically targeting cell structures and / or melanin at low temperatures, preferably below 0°C, more preferably at a temperature of -90°C to -10°C.

[0240] According to one embodiment, the embedding agent and / or imaging material as defined above is useful for specifically targeting antibodies, preferably antibodies coupled to nanoscale gold nanoparticles. According to one embodiment, the embedding agent and / or imaging material as defined above is useful for specifically targeting antibodies coupled to nanoscale gold nanoparticles by aggregating silver nitrate embedded in the embedding agent and / or imaging material. According to one embodiment, the embedding agent and / or imaging material as defined above is useful for producing a silver enhancement reaction at low temperatures, preferably at a temperature below 0°C.

[0241] Reagent test kit

[0242] The invention also relates to a kit for preparing a biological sample to be imaged, preferably by microscopy.

[0243] According to one embodiment, the kit comprises a first container and a second container, the first container comprising at least one monomer as defined above; the second container comprising an initiator. According to one embodiment, the kit comprises a first container and a second container, the first container comprising a hydrophilic monomer mixture as defined above; the second container comprising an initiator.

[0244] According to one embodiment, the kit comprises:

[0245] - a first container comprising (a) an alkylene glycol dimethacrylate and (b) a polyalkylene glycol diacrylate or a polyalkylene glycol methacrylate, the polyalkylene glycol diacrylate or the polyalkylene glycol methacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group;

[0246] - A second container comprising a free radical polymerization initiator.

[0247] According to one embodiment, the kit comprises:

[0248] - a first container comprising (a) triethylene glycol dimethacrylate; and (b) poly(propylene glycol) diacrylate; and

[0249] - A second container comprising benzoin methyl ether or hydroxy-2-methylpropiophenone.

[0250] According to one embodiment, the invention also relates to a kit comprising a container comprising at least one monomer and an initiator as defined above, and a mixing device.

[0251] According to one embodiment, the present invention also relates to a kit comprising:

[0252] - a container comprising (a) an alkylene glycol dimethacrylate, (b) a polyalkylene glycol diacrylate or a polyalkylene glycol methacrylate, the polyalkylene glycol diacrylate or the polyalkylene glycol methacrylate being optionally substituted with at least one hydrophilic group, such as a hydroxyl, amino or carbonyl group; and (c) a free radical polymerization initiator; and

[0253] -Mixing device.

[0254] According to one embodiment, the mixing device is such as, but not limited to, a container, a capsule, a mixer and / or a spatula. BRIEF DESCRIPTION OF THE DRAWINGS

[0255] Figure 1 is obtained by transmission electron microscopy (TEM) and embedded in the embedding agent of the present invention ( Figure 1 A) or Resin( Figure 1 B) Negative photograph of HeLa cells.

[0256] Figure 2 The negative film of HeLa cells embedded in the embedding medium of the present invention was obtained by focused ion beam scanning electron microscopy (FIB-SEM).

[0257] Figure 3 is a comparison of two scanning electron microscopy (SEM3View) images of the imaging material of the present invention containing Caenorhabditis elegans, using 0.01% uranyl acetate for contrast during freeze substitution. A is embedded in the embedding medium of the present invention, and B is embedded in The first negative shows a sharp and even contrast. The second negative shows a large amount of charge as white spots, the square areas used to focus the negative. The electrons accumulate on the surface of the squares and cannot be clearly imaged.

[0258] Figure 4elegans embedded in the embedding medium of the present invention. Film 4A is obtained by scanning electron microscopy (SEM); film 4B is obtained by green fluorescent protein (GFP); film 4C is obtained by red fluorescent protein (RFP); film 4D is obtained by "transmitted light"; film 4E is a multi-channel image.

[0259] Figure 5 yes Figure 4 Magnified images of fluorescence microscopy films. Film 5A was obtained by scanning electron microscopy (SEM); film 5B was obtained by green fluorescent protein (GFP); film 5C was obtained by red fluorescent protein (RFP); and film 5D is a multi-channel image. Example

[0260] The present invention will be explained in more detail with reference to the following examples. These examples are intended to represent specific embodiments of the present invention and are not intended to limit the scope of the invention.

[0261] abbreviation

[0262] ℃: Celsius

[0263] CDCl3: deuterated chloroform

[0264] CLEM: Correlative Light and Electron Microscopy

[0265] Eq: equivalent

[0266] PEG: polyethylene glycol

[0267] PPGdA: Poly(propylene glycol) diacrylate

[0268] g: grams

[0269] h: hours

[0270] mmol: millimole

[0271] mol: mole

[0272] NMR: Nuclear Magnetic Resonance

[0273] TGdMA: Triethylene glycol dimethacrylate

[0274] UV: Ultraviolet

[0275] Materials and methods

[0276] Reagents, solvents, and starting materials were purchased from Sigma Aldrich and used without further purification.

[0277] UV irradiation

[0278] UV irradiation of the samples was carried out by using a UV lamp system F300S Heraeus from Fusion Fusion. Or Leica AFS-1 or AFS-2 for freeze substitution protocol (automated freeze substitution is performed automatically).

[0279] Part I - Preparation of embedding medium

[0280] Example 1: Synthesis of poly(propylene glycol) diacrylate (PPGdA)

[0281] Synthesis of PPGdA by acryloylation reaction: 800 g / mol (20 g, 25 mmol) of poly(propylene glycol) and triethylamine (11.7 g, 115 mmol) were added to a round-bottom flask filled with 140 mL of dichloromethane. The mixture was cooled to 5 °C and acryloyl chloride (9.5 g, 105 mmol) was added dropwise under magnetic stirring.

[0282] Depending on the molecular weight of the poly(propylene glycol) used, the ratio of trimethylamine and acryloyl chloride has to be adjusted (2.3 equivalents and 2.1 equivalents per hydroxyl function, respectively).

[0283] After 2 hours, the reaction was complete. The triethylamine salt was filtered off and the product was washed several times with alkaline, acidic and neutral water. The organic phase was dried over anhydrous sodium sulfate and filtered. After the solvent was removed under reduced pressure, the final product (20.7 g, yield: 91%) was obtained.

[0284] 1 H NMR (CDCl3, 300MHz): 6.43-6.35 (2H, d, H-CH=CH-); 6.15-3.06 (2H, d, H-CH=CH-), 5.81-5.78 (2H, d, H-CH=CH-), 5.18-5.02 (2H, m, -C ( O)-O-CH-CH2-), 3.83-3.18(42H, m, -CH(CH3)-CH2-), 3.83-3.18(42H, m, -CH(CH3)-CH2-O-), 1.39-0.99(42H, m, -CH(CH3)-CH2-O-).

[0285] Example 2: Preparation of the embedding agent of the present invention

[0286] 2.1. One-pot method

[0287] Preparation 1

[0288] Triethylene glycol dimethacrylate (TGdMA), polypropylene glycol diacrylate (PPGdA) and an initiator, preferably benzoin methyl ether and / or hydroxy-2-methylpropiophenone, are added to a flask in a mass ratio of 79 / 20 / 1 and stirred at room temperature. The final embedding medium 1 is stored away from light and / or heat.

[0289] Preparation 2

[0290] Triethylene glycol dimethacrylate (TGdMA) and an initiator, preferably benzoin methyl ether and / or hydroxy-2-methylpropiophenone, are added to the flask in a mass ratio of 99 / 1 and stirred at room temperature. The final embedding medium 2 is stored away from light and / or heat.

[0291] Preparation 3

[0292] Triethylene glycol dimethacrylate (TGdMA), hydroxy PEG methacrylate and an initiator, preferably benzoin methyl ether and / or hydroxy-2-methylpropiophenone, are added to the flask in a mass ratio of 74 / 25 / 1 and stirred at room temperature. The final embedding medium 3 is stored away from light and / or heat.

[0293] Preparation 4

[0294] Triethylene glycol dimethacrylate (TGdMA), polypropylene glycol diacrylate (PPGdA), silver nitrate solution (1.4 mM) and an initiator, preferably benzoin methyl ether and / or hydroxy-2-methylpropiophenone, are added to a flask in a mass ratio of 72.4 / 18.1 / 9 / 0.5 and stirred at room temperature. The final embedding medium 4 is stored away from light and / or heat.

[0295] 2.2. Kits containing the embedding agent of the present invention

[0296] Triethylene glycol dimethacrylate (TGdMA) and poly(propylene glycol) diacrylate (PPGdA) were added to the first container at a mass ratio of 79 / 20 and stirred at room temperature.

[0297] In a second container is stored an initiator, preferably benzoin methyl ether and / or hydroxy-2-methylpropiophenone.

[0298] After mixing the contents of the first container and the second container to obtain a matrix precursor solution, the mixture is preferably kept away from light and / or heat.

[0299] Part II - Preparation of conductive imaging materials and electron microscopy imaging

[0300] Example 3: Method of preparing biological samples for microscopic analysis

[0301] The purpose is to embed biological samples to provide fluorescent and / or conductive biological imaging materials for electron microscopy. To this end, the applicant used the embedding medium 1 of Example 2 for processing.

[0302] General approach

[0303] Step 1 - Vitrification and / or Fixation / Dehydration of Biological Samples

[0304] First, the biological sample is vitrified by any cryogenic method known to the skilled person, and then a solvent is added at -90°C, which may be ethanol, methanol or acetone (the biological sample in the solvent is referred to as a "freeze substitution mixture"). Optionally, a chemical fixative and / or a contrast agent may be added. Examples of chemical fixatives may be osmium, osmium tetroxide, uranyl acetate, glutaraldehyde and paraformaldehyde. Examples of contrast agents may be uranyl acetate, lead derivatives, silver derivatives, osmium derivatives or tannic acid.

[0305] The biological sample is kept at -90°C for a period ranging from 1 minute to several days. Then, the temperature is slowly raised to -40°C, and the freeze substitution mixture is removed by continuous washing with pure solvents. A dehydrated biological sample is obtained.

[0306] Step 2 - Infiltration of embedding medium 1

[0307] Next, the dehydrated biological sample obtained in step 1 is sequentially infiltrated with a mixture of an embedding agent / solvent until it is infiltrated with only the embedding agent solution, thereby obtaining a composition comprising a dehydrated biological sample embedded in the matrix precursor solution of the present invention.

[0308] Step 3 - Preparation of Polymerization-Conductive Bioimaging Materials

[0309] The composition obtained in step 2 is then polymerized by photopolymerization or by thermal polymerization.

[0310] In the case of photopolymerization, the composition is initiated at -40°C by UV irradiation for 24 to 48 hours. Then, the temperature is slowly raised to room temperature, always under UV irradiation. An additional 24 hours of irradiation is performed at room temperature to achieve complete polymerization inside the biological sample.

[0311] An imaging material that is conductive and retains fluorescence is obtained.

[0312] Step 4b (optional) - microtomy or ultramicrotomy

[0313] Conductive imaging materials are optionally prepared by microtome and / or ultrathin sectioning and then observed by electron microscopy.

[0314] Optional additional steps - optical microscopy

[0315] In this method, the conductive imaging material may be subjected to optical microscopy analysis in any one of steps 1 to 4 before being observed by electron microscopy.

[0316] Part III - Electron Microscopy Observation

[0317] Example 4: Microscopic analysis

[0318] To demonstrate the superiority of the imaging material of the present invention when used in electron microscopy, biological samples were prepared according to the protocol described in Example 3, wherein the freezing mixture contained 0.05% uranyl acetate, 0.01% glutaraldehyde, 1% water, and was performed at a temperature of -90°C to -45°C.

[0319] Imaging

[0320] The purpose is to compare with The increase in contrast obtained by the embedding medium of the present invention on HeLa cells compared to HM 20 resin, which used the same freeze substitution protocol (i.e. using a freezing mixture containing 0.05% uranyl acetate, 0.01% glutaraldehyde and 1% water at a temperature of -60°C to -45°C).

[0321] HeLa cells were observed by transmission electron microscopy (TEM).

[0322] Figure 1 Display, with HM 20 resin( Figure 1 B) compared to the material obtained, the material of the present invention (see Figure A) achieves better contrast.

[0323] Analysis of HeLa cells embedded in the material of the present invention was also observed by scanning electron microscopy (SEM). Figure 2 It was shown that the material of the present invention enables fine visualization of the structure of cells embedded inside the material.

[0324] Targeting melanin

[0325] In this experiment, MNT1 cells (immortalized melanocyte cell line, MNT1 line) were studied.

[0326] The imaging material of the present invention comprising melanosomes and silver salt aggregates was observed by electron microscopy.

[0327] Unexpectedly, the corresponding negatives ( Figure 3 A) shows that silver salts accumulate specifically in biological structures.

[0328] Without being bound by any theory, the applicant believes that during microscopic studies a specific reaction occurs between the silver salt incorporated into the imaging material of the invention and the melanin aggregated into the amyloid fibrils of the melanosomes.

[0329] To demonstrate this specific reaction, the same experiment was performed by replacing melanosomes with HeLa cells that do not express melanin.

[0330] The corresponding negatives ( Figure 3 B) shows that the silver salt is dispersed in the material but is not specifically localized in the biological sample.

[0331] Therefore, the imaging material of the present invention, particularly the embedding agent, when comprising a silver salt, can specifically target biological samples containing melanin.

[0332] Fluorescence preservation

[0333] The preserved fluorescence of MNT1 cells in the imaging material was analyzed by structured illumination microscopy (SIM), a light microscopy technique.

[0334] Figure 4 and Figure 5 It was shown that the internal fluorescence of HeLa cells can be effectively detected from the imaging material of the present invention.

Claims

1. A conductive embedding agent for a biological sample, comprising: - 60 to 99% by weight of a glycol dimethacrylate selected from the group consisting of ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, propylene glycol dimethacrylate and butylene glycol dimethacrylate; - from greater than 0% to 38% by weight of a polyalkylene glycol diacrylate selected from polyethylene glycol diacrylate, polypropylene glycol diacrylate and polybutylene glycol diacrylate; and - 0.1 to 2% by weight of a free-radical polymerization initiator.

2. The conductive embedding agent according to claim 1, further comprising at least one additive.

3. The conductive embedding agent according to claim 2, wherein the at least one additive comprises at least one heavy metal salt or lanthanide salt. The conductive embedding agent according to claim 1 , wherein the glycol dimethacrylate is triethylene glycol dimethacrylate. The conductive embedding agent according to claim 1 , wherein the polyalkylene glycol diacrylate is polypropylene glycol diacrylate.

6. The conductive embedding agent according to claim 1 or claim 2, comprising: - 60 to 99% by weight of triethylene glycol dimethacrylate; - from greater than 0% to 38% by weight of polypropylene glycol diacrylate; and - 0.1 to 2% by weight of a free-radical polymerization initiator.

7. The conductive embedding agent according to claim 1 or claim 2, comprising: - 79% by weight of triethylene glycol dimethacrylate; - 20% by weight of polypropylene glycol diacrylate; and - 1% by weight of benzoin methyl ether.

8. The conductive embedding agent according to claim 3, wherein the at least one heavy metal salt comprises osmium acetate and / or uranyl acetate.

9. The conductive embedding agent according to claim 1 or claim 2, further comprising at least one biological sample.

10. The conductive embedding agent according to claim 9, wherein the at least one biological sample is a vitrified and / or chemically fixed and / or dehydrated biological sample.

11. A conductive imaging material comprising at least one biological sample embedded in a polymer matrix obtained by polymerization of the conductive embedding agent according to any one of claims 1 to 10.

12. The conductive imaging material according to claim 11, which retains the intrinsic fluorescence, protein activity and / or antigen specificity of the embedded biological sample.

13. A non-diagnostic method for imaging a biological sample by microscopy, comprising the steps of: (i-1) fixing and / or dehydrating the biological sample; (ii) infiltrating the biological sample of step (i-1) with the conductive embedding agent according to any one of claims 1 to 10; (iii) polymerizing the mixture obtained in step (ii) to obtain a conductive imaging material; (iv) imaging the conductive imaging material of step (iii) by optical microscopy and / or electron microscopy.

14. The method according to claim 13, further comprising the step of performing sectioning or ultra-thin sectioning on the conductive imaging material.

15. Non-diagnostic use of a conductive embedding agent according to any one of claims 1 to 10 or a conductive imaging material according to claim 11 or 12 for imaging a biological sample by microscopy.

16. Use according to claim 15, wherein the microscopy technique is optical and / or electron microscopy.

17. A kit for preparing a biological sample to be imaged by microscopy, comprising: - a first container comprising (a) 60 to 99% by weight of a glycol dimethacrylate selected from the group consisting of ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, propylene glycol dimethacrylate, and butylene glycol dimethacrylate; and (b) greater than 0 to 38% by weight of a polyalkylene glycol diacrylate selected from the group consisting of polyethylene glycol diacrylate, polypropylene glycol diacrylate, and polybutylene glycol diacrylate; - a second container comprising a free radical polymerization initiator; or contain: A container comprising (a) 60 to 99 weight percent of a glycol dimethacrylate selected from the group consisting of ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, propylene glycol dimethacrylate, and butylene glycol dimethacrylate; (b) greater than 0 to 38 weight percent of a polyalkylene glycol diacrylate selected from the group consisting of polyethylene glycol diacrylate, polypropylene glycol diacrylate, and polybutylene glycol diacrylate; and (c) 0.1 to 2 weight percent of a free radical polymerization initiator; and mixing devices.

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