Composition and method for clearing biological samples

Abstract

The present invention relates to a composition for clearing biological samples, at least comprising: - a delipidation and permeabilization agent; - a refractive index homogenizing agent; the delipidation and permeabilization agent being a compound selected from among terpenes or terpenoids, preferably terpenes.

Description

[0001] COMPOSITION AND METHOD FOR TRANSPARING BIOLOGICAL SAMPLES

[0002] Field of invention

[0003] The present invention relates to a composition for transparing biological samples and its use for making a biological sample transparent. More particularly, the present invention relates to a transparing composition and a method for rapid and non-toxic transparing of biological samples.

[0004] Technical background

[0005] The study of the three-dimensional structure of biological samples such as tissues and organs gives rise to the need for volumetric, or 3D, optical imaging to acquire complete structural information. However, optical imaging of thick tissues or whole organs is limited by the autofluorescence of the biological sample, light absorption and more particularly by light scattering.

[0006] Autofluorescence has the effect of erasing the contrast between the background and the specific marking, the specific marking then merges with the intrinsic fluorescence coming from the sample.

[0007] Light absorption has the effect of reducing the number of photons exponentially with the thickness of the biological sample. The effect is then twofold in fluorescence imaging because, (i) the fluorescence excitation light does not reach the entirety of a thick sample, and (ii) the absorption also attenuates the signal created, the fluorescence before being collected by the objective.

[0008] Finally, light scattering has the effect of spatially redistributing photons in the sample, resulting in heterogeneity of the refractive indices composing the biological sample. However, imaging by (conventional) optical microscopy typically relies on the spatial detection of photons not deviated from their initial trajectories. Scattering therefore reduces the depth of imaging techniques to a few tens of pm. Consequently, imaging of biological tissues is fundamentally limited by light scattering.

[0009] Major advances in the field have been made thanks to the development of techniques for making biological samples transparent, which are based in particular on the homogenization of refractive indices in the biological sample in order to reduce homogeneity defects in light scattering and allow the observation of deeper structures in the biological sample.

[0010] However, these techniques have in common their slowness, often of the order of several hours or days, depending on the size of the sample transparent, reducing the number of samples that can be analyzed per day, or even per week, and immobilizing material and human resources.

[0011] Additionally, these techniques include solvent-based clarification methods and involve the handling of hazardous reagents. For example, methyl salicylate, benzyl alcohol, benzyl benzoate, dichloromethane, and dibenzyl ether are used as clearing compositions (Becker et al. (2012) Pios one, 7: 633916).

[0012] Thus, despite the significant progress made in the field, no technique is satisfactory and there is a need for alternative methods for transparentizing biological samples that are faster, less toxic and offer good transparency performance. In addition, current methods that work well often do so on one type of sample only; when the preparation or the animal model changes, everything remains to be redone, optimized, and modified in order to obtain satisfactory results. Thus, there is a need, particularly for microscopy services and imaging platforms, for a more generally applicable method.

[0013] Summary of the invention

[0014] The present invention arises from the unexpected discovery by the inventors that a transparizing composition comprising a terpene, in particular D-limonene, and thiodiethanol (TDE) in a concentration of 60% makes it possible to obtain maximum transparizing of biological samples more quickly than the transparizing obtained with conventional transparizing compositions.

[0015] The inventors have demonstrated that the method for making biological samples transparent using a transparent composition as defined above is very rapid, has good transparent performance, is non-toxic for the handler and non-corrosive for the equipment used, in particular the microscope objective.

[0016] In addition, the biological sample made transparent by the method or the transparency composition according to the invention retains its morphology, its volume and its fluorescence.

[0017] Furthermore, the inventors have demonstrated that the transparency method using the transparency composition defined above has a universal character because it applies to the transparency of thick biological samples such as those of mammals, insects, plants, and small fish.

[0018] Thus, the present invention relates to a composition for making biological samples transparent, comprising at least:

[0019] - a defatting and permeabilizing agent;

[0020] - a refractive index homogenizing agent; wherein the delipidation and permeabilization agent is a compound selected from the group consisting of terpenes and terpenoids, preferably terpenes. The present invention also relates to the use of a transparency composition as defined above, for making a biological sample transparent.

[0021] The present invention also relates to a method for making a biological sample transparent comprising a step of bringing a biological sample into contact with a transparency composition as defined above.

[0022] The present invention also relates to a kit for making a biological sample transparent comprising:

[0023] - a composition comprising a defatting and permeabilizing agent, such as D-limonene,

[0024] - a composition comprising a refractive index homogenizing agent, such as thiodiglycol (TDE),

[0025] - optionally, a depigmentation composition comprising H202, an alcohol and a chelating agent, such as citrates,

[0026] - optionally, a demineralization solution comprising a divalent ion chelator, such as EDTA, BAPTA or citrate.

[0027] Detailed description of the invention

[0028] Definitions

[0029] The term "comprising" means "including", "containing" or "encompassing", that is, when an object "comprises" one or more elements, other elements than those mentioned may also be included in the object. Conversely, the expression "consisting of" means "made up of", that is, when an object "consists of" one or more elements, the object cannot include elements other than those mentioned.

[0030] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0031] Unless otherwise stated, percentage (%) values ​​refer to volume / volume (v / v) percentages.

[0032] "Clearing" or "clarifying" means making biological samples transparent to light, typically in the visible band, but generally from UV to IR.

[0033] The terms “biological sample,” “clinical sample,” and “sample” may be used interchangeably and refer to human or animal tissues, organs, cells, insects, whole animals, and plants, as well as fragments thereof such as slices, pieces, or sections.

[0034] The terms "tissues" and "organs" include soft tissues, hard tissues, calcified tissues, and tissues from genetically modified organisms. As an example of a biological sample according to the invention, it is possible to cite the heart, blood vessels, salivary glands, esophagus, stomach, liver, gallbladder, pancreas, intestine, colon, rectum, endocrine glands, adrenal glands, kidneys, ureter, bladder, lymph node, tonsils, spleen, skin, muscles, brain, spinal cord, nerves, ovaries, uterus, vagina, mammary gland, testicles, prostate, pharynx, larynx, trachea, bronchi, lungs, diaphragm, cartilage, ligaments, tendons, bones, teeth, fish scales, fish fins, crustaceans, insects such as a wasp, a grasshopper, a spider, a fly, a policeman, a bedbug etc.plants such as a leaf, a root, a stem, a petal, a flower, etc., whole animals such as mice, as well as fragments of these.

[0035] Preferably, the biological sample according to the invention has a size of between 5 μm and 10 cm. Preferably, the biological sample according to the invention has a size of less than 10 cm, 9 cm, 8 cm, 7 cm, 6 cm, 5 cm, 4 cm, 3 cm, 2 cm, 1 cm. Also preferably, the biological sample according to the invention has a size of greater than 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm. More preferably, the biological sample according to the invention has a size of between 1 mm and 5 cm, between 1 mm and 2 cm, between 1 mm and 1 cm. Preferably, the biological sample according to the invention has a size of approximately 1 mm, 5 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, 16 mm, 17 mm, 18 mm, 19 mm, 20 mm.

[0036] The terms "labeling agent," "dye," "labeling material," and "probe" can be used interchangeably and refer to any material capable of targeting a specific molecule on a biological sample. These can be chemical compounds or biological compounds.

[0037] The terms "container," "receptacle," and "tube" can be used interchangeably.

[0038] Transparency composition

[0039] Preferably, the delipidation and permeabilization agent according to the invention is selected from the group consisting of terpenes and terpenoids.

[0040] Preferably, the delipidation and permeabilization agent according to the invention is a terpene selected from the group consisting of the monoterpene limonene, farnesol and nerolidol.

[0041] More preferably, the defatting and permeabilizing agent according to the invention is a monoterpene limonene. Even more preferably, the defatting and permeabilizing agent according to the invention is D-limonene. D-limonene, also known as (4R)-i-methyl-4-prop-i-en-2- ylcyclohexene or (R)-(+)-limonene or (+)-limonene is the dextrorotatory R-enantiomer of the monoterpene limonene and is well known to those skilled in the art.

[0042] Farnesol, also known as (2E,6E)-3,7,ii-trimethyldodeca-2,6,io-trien-i-ol, is well known to those skilled in the art.

[0043] Nerolidol, also known as 3,7,n-trimethyldodeca-i,6,io-triene-3-ol, is well known to those skilled in the art.

[0044] Preferably, the transparizing composition according to the invention comprises from 0.5% to 20% volume / volume of delipidation and permeabilization agent, in particular terpene, relative to the total volume of the transparizing composition. Preferably, the transparizing composition according to the invention comprises from 0.5% to 15%, from 0.5% to 10%, from 0.5% to 9%, from 0.5% to 8%, from 0.5% to 7%, from 0.5% to 6%, from 0.5% to 5%, from 0.5% to 4%, from 0.5% to 3%, from 0.5% to 2%, from 0.5% to 1.5%, from 0.5% to 1%, advantageously approximately 1% volume / volume of delipidation and permeabilization agent, in particular terpene, relative to the total volume of the transparizing composition.

[0045] Preferably, the refractive index homogenizing agent according to the invention is selected from the group consisting of thiodiglycol (TDE), ethyl cinnamate and sucrose. More preferably, the refractive index homogenizing agent is thiodiglycol (TDE).

[0046] Preferably, the transparizing composition according to the invention comprises from 50% to 90%, for example 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50 to 60% preferably, 55% to 80%, 55% to 70%, 55 to 65% volume / volume of refractive index homogenizing agent, in particular thiodiglycol, relative to the total volume of the transparizing composition.

[0047] More preferably, the transparizing composition according to the invention comprises 60% volume / volume of refractive index homogenizing agent, in particular thiodiglycol, relative to the total volume of the transparizing composition.

[0048] Preferably, the transparency composition according to the invention further comprises at least one non-ionic surfactant.

[0049] Preferably, the non-ionic surfactant according to the invention is present in the composition in a concentration of between 0.5 and 1%.

[0050] As an example of a non-ionic surfactant according to the invention, it is possible to cite esters of fatty acids and polyoxyethylene sorbitan, also known as polysorbates, such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate and polyoxyethylene sorbitan monooleate, alcohols such as polyvinyl alcohol and alkylphenols such as polyoxyethylene octylphenyl ether, and NP-40 (Nonyl phenoxypolyethoxylethanol). Preferably, the nonionic surfactant according to the invention is selected from the group consisting of Triton X such as Triton X-100 and Triton X-140, polysorbates such as polysorbate 20 (Tween-20), polysorbate 40 (Tween-40), polysorbate 60 (Tween-60), and polysorbate 80 (Tween-80) and mixtures thereof.

[0051] More preferably, the non-ionic surfactant according to the invention is selected from the group consisting of Triton X-100, polysorbate 80 (Tween-80) and polysorbate 20 (Tween-20).

[0052] Even more preferably, the non-ionic surfactant according to the invention is Triton X-100, preferably in a concentration of 0.5 to 1% in the composition according to the invention.

[0053] Preferably, the transparency composition according to the invention further comprises at least one agent delaying the alteration of fluorescence by light, also known as an antifading agent. The agent delaying the alteration of fluorescence according to the invention may be chosen from any agent delaying the alteration of fluorescence well known to those skilled in the art. As examples of agents delaying the alteration of fluorescence which may be used in the transparency composition according to the invention, it is possible to cite DABCO (also known as triethylenediamine or its acronym, TEDA), paraphenylenediamine (PPD), n-propylgalate.

[0054] The transparizing composition according to the invention may contain other additional additives well known to those skilled in the art. As examples of additional additives that may be used in the transparizing composition according to the invention, it is possible to cite solubilizing agents, diluents, emulsifying agents, preservatives, lubricants, thickening agents, gelling agents, pH adjusting agents, osmotic pressure controlling agents, etc.

[0055] Preferably, the transparency composition according to the invention is in the form of an aqueous composition. The composition according to the invention may comprise an aqueous medium such as acid solutions and buffer solutions such as PBS and Tris-HCl.

[0056] Preferably, the transparency composition according to the invention has a pH between 5 and 9, between 5.5 and 8.5, between 6 and 8. The pH of the transparency composition according to the invention can be adjusted by a person skilled in the art.

[0057] According to a particular embodiment of the invention, the transparency composition comprises:

[0058] - D-limonene as a defatting and permeabilizing agent;

[0059] - a refractive index homogenizing agent;

[0060] - a non-ionic surfactant, preferably triton X-100. Preferably, the refractive index homogenizing agent is TDE. More preferably, the refractive index homogenizing agent is TDE in an amount of 60% volume / volume relative to the total volume of the transparency composition.

[0061] According to a particular embodiment of the invention, the transparency composition comprises another terpene such as farnesol as a delipidation and permeabilization agent, and a refractive index homogenizing agent. Preferably, the refractive index homogenizing agent is TDE in an amount of 60% volume / volume relative to the total volume of the composition.

[0062] Use

[0063] Preferably, the transparency composition according to the invention is used to make a biological sample according to the invention transparent.

[0064] Preferably, the transparency composition according to the invention is used to make a biological sample transparent, in particular a soft tissue, a hard tissue, a calcified tissue, a tissue from a genetically modified organism, an insect, an animal, a plant, a cell, a fish, as well as fragments of these.

[0065] The transparency composition according to the invention can be used in biology applications for scientific research (neurosciences, organoids, developmental biology, etc.) taking into account the size of the samples, medical histology (analysis of biopsies, extemporaneous specimens), forensic medicine, anatomy, field medical diagnosis (developing countries), marine biology, entomology, research in plant biology, in agriculture, in agri-food, teaching for amateurs or naturalists, play applications for children, in the arts, in biomimicry, in architecture, for museums and collections, etc.

[0066] The transparency composition according to the invention can be used in biological imaging, in particular in fluorescence imaging. Preferably, the transparency composition according to the invention is used for imaging the autofluorescence or fluorescence of a soft tissue, a hard tissue, a calcified tissue, a tissue from a genetically modified organism, an insect, an animal, a plant, a cell as well as fragments thereof.

[0067] The transparency composition according to the invention can also be used in immunofluorescence.

[0068] Process

[0069] Preferably, the method for making a biological sample transparent according to the invention comprises a step of bringing the biological sample according to the invention into contact with a transparency composition according to the invention for a time sufficient to make the biological sample transparent. Preferably, the transparency step according to the invention is carried out in a single step.

[0070] Preferably, the biological sample made transparent by the transparency composition according to the invention and / or by the method according to the invention retains its volume and its morphology. When making a biological sample transparent according to the invention, a reduction in the size of the biological sample of between 1 and 25%, for example between 5% and 20%, between 10% and 20%, between 15% and 20%, preferably of approximately 20% compared to the initial size of the biological sample can be observed.

[0071] Also preferably, the fluorescence of most of the fluorophores and fluorescent proteins of the biological sample is preserved when it is transparentized.

[0072] The time for bringing the biological sample into contact with the transparency composition according to the invention depends on the type of biological sample used and its size. The time for bringing the biological sample into contact with the transparency composition according to the invention necessary to make the biological sample transparent can easily be determined by a person skilled in the art.

[0073] Preferably, the time of contact of the biological sample with the transparency composition is between 48 hours and 30 seconds, preferably between 24 hours and 30 seconds, 24b and 1 minute, 2oh and 1 minute, 15I1 and 1 minute, 10 and 1 minute, 5I1 and 1 minute, 5I1 and 30 minutes, 5h and 20 minutes, 1h and 20 minutes.

[0074] Preferably, the time of contact of the biological sample with the transparency composition according to the invention is less than 48 hours, less than 42 hours, less than 40 hours, less than 30 hours, less than 24 hours, less than 20 hours, less than 15 hours, less than 10 hours, less than 5 hours, less than 1 hour. More preferably, the time of contact of the biological sample with the transparency composition according to the invention is less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 12 minutes, less than 10 minutes.

[0075] The time of contact of the biological sample with the transparency composition may be greater than 48 hours, for example in the case of large biological samples such as a whole animal such as a whole mouse.

[0076] For example, when the biological sample intended to be transparentized is a soft tissue such as, for example, the brain, lung, heart, liver, kidney, spleen, testicle, spinal cord, blood vessels, skin, intestines, human or animal or fragments thereof, having a size preferably less than 2 cm, the time of contact of the biological sample with the transparentizing composition is between 1 hour and 1 minute, preferably between 20 minutes and 1 minute, more preferably between 10 minutes and 1 minute. For example, the contact time of the biological sample with the transparentizing composition according to the invention is between 48 hours and 1 hour when the biological sample intended to be transparentized is an insect such as, for example, a grasshopper, a gendarme, a bedbug, a spider, etc.

[0077] Preferably, the temperature of contacting the biological sample according to the invention with the transparency composition according to the invention is between 10°C and 60°C, preferably between 15°C and 60°C, between 18°C ​​and 55°C, between 23°C and 50°C, between 25°C and 45°C, more preferably between 30°C and 42°C and even more preferably between 37°C and 40°C.

[0078] When the contacting of the biological sample with the transparency composition according to the invention is carried out at room temperature, i.e. between 20 and 25°C, or at a higher temperature, preferably between 37°C and 42°C, the time required to make the biological sample transparent decreases compared to transparency carried out at a temperature below 20°C, preferably below 10°C.

[0079] Any type of equipment well known to those skilled in the art can be used to heat the transparizing composition according to the invention. As an example of equipment that can be used to heat the transparizing composition according to the invention, preferably between 20 and 42°C, more preferably at approximately 37°C, it is possible to cite a water bath or an oven.

[0080] The method according to the invention may also comprise a step of fixing the biological sample before bringing the biological sample into contact with the transparency composition according to the invention. Any method of fixing a biological sample well known to those skilled in the art may be used to fix the biological sample according to the invention. By way of example, the biological sample according to the invention may be fixed by a fixing agent or a mixture of fixing agents, for example by immersing the biological sample in a solution of fixing agent at a temperature of approximately 4°C and then rinsing with a physiological solution or by a perfusion technique. As examples of fixing agents that may be used, it is possible to cite aldehydes, such as formalin and paraformaldehyde, alcohols, such as ethanol and methanol, acids such as acetic acid, glyoxal, and mixtures thereof.Preferably, the fixed biological sample may be stored at 4°C for up to 1 year, preferably in the presence of a physiological buffer such as PBS and a bacterial and fungal culture inhibitor such as sodium azide.

[0081] The method according to the invention may also comprise a step of dehydrating the biological sample. This step is optional and may be carried out according to conventional biological sample dehydration methods well known to those skilled in the art. For example, it is possible to dehydrate the biological sample according to the invention by replacing the water it contains with alcohol such as ethanol, for example by immersing the sample in a succession of alcohol baths of increasing concentration.

[0082] The method according to the invention may also comprise a step of including the biological sample in a hydrogel such as, for example, agarose or phytagel. Advantageously, the inclusion step is intended to facilitate the handling of the biological sample. Preferably, the inclusion of the biological sample is carried out after the step of demineralization, depigmentation and optionally immunofluorescence of the biological sample. Preferably, the inclusion of the biological sample is carried out before the step of bringing the biological sample into contact with the transparency composition, more preferably, between the depigmentation step and the transparency step when a depigmentation step is necessary. The inclusion of the biological sample may be carried out according to conventional inclusion methods well known to those skilled in the art. For example, the sample may be deposited oriented in a mold comprising hydrogel.Once the gel has set, at room temperature or 4°C, the sample is removed from the mold.

[0083] The biological sample embedded in the hydrogel can be cut into thin section slices. Preferably, the thickness of the slices is less than 5 cm, for example less than 4 cm, less than 3 cm, less than 2 cm, less than 1.5 cm, less than 1 cm. More preferably, the thickness of the slices is less than 100 mm, less than 90 mm, less than 80 mm, less than 70 mm, less than 60 mm, less than 50 mm, less than 40 mm, less than 30 mm, less than 20 mm, less than 10 mm, less than 9 mm, less than 8 mm, less than 7 mm, less than 6 mm, less than 5 mm, less than 4 mm, less than 3 mm, less than 2 mm, less than 1 mm.

[0084] Preferably, the thickness of the slices is between 5 pm and 5 cm, more preferably between 10 pm and 2 cm, 20 pm and 1 cm, 30 pm and 1 cm, 40 pm and 1 cm, 60 pm and 1 cm, 70 pm and 1 cm, 80 pm and 1 cm, 90 pm and 1 cm, 100 pm and 1 cm, 200 pm and 1 cm, 300 pm and 1 cm, 400 pm and 1 cm, 500 pm and 1 cm.

[0085] More preferably, the thickness of the slices is between 5 pm and 10 mm, 5 pm and 9 mm, 5 pm and 8 mm, 5 pm and 7 mm, 5 pm and 6 mm, 5 pm and 5 mm, 5 pm and 4 mm, 5 pm and 3 mm, 5 pm and 2.5 mm, 5 pm and 2 mm, 5 pm and 1.5 mm, 5 pm and 1 mm.

[0086] The method according to the invention may also comprise a step of immobilizing the biological sample on a surface. The immobilization of the biological sample may be carried out according to any sample immobilization method well known to those skilled in the art. As an example of a surface that may be used, it is possible to cite a slide, a plate, a well, a tube, a membrane or a film.

[0087] The method according to the invention may also comprise a step of depigmenting the biological sample before the step of bringing the biological sample into contact with the transparency composition according to the invention. Preferably, the step of depigmenting the biological sample is carried out in order to improve the depigmentation of highly colored tissues such as the liver, kidneys, lungs, etc. Those skilled in the art can easily determine when a depigmentation step is necessary. The depigmentation step according to the invention is preferably carried out in a single step.

[0088] The contact time of the biological sample with the pretreatment composition varies depending on the type and size of the biological sample and can be easily determined by a person skilled in the art. For example, the contact time of the biological sample with the pretreatment composition can range from a few minutes for small samples to several days for insects or fish. Preferably, the contact time of the biological sample with the pretreatment composition is between 10 minutes and 24 hours.

[0089] The depigmentation step according to the invention is preferably carried out at a temperature between 20°C and 42°C, preferably at approximately 37°C.

[0090] Preferably, the step of depigmenting the biological sample is carried out by contacting the biological sample with a composition, also called a pretreatment composition, comprising hydrogen peroxide (H202), an alcohol, and a chelating agent and a buffer.

[0091] Preferably, the alcohol of the pretreatment composition according to the invention is selected from the group consisting of primary and secondary alcohols. More preferably, the alcohol is selected from methanol and ethanol. Even more preferably, the alcohol according to the invention is ethanol.

[0092] Preferably, the chelating agent of the pretreatment composition according to the invention is a divalent ion chelator. Preferably, the chelating agent of the pretreatment composition according to the invention may be EDTA (ethylenediaminetetraacetic acid), but is preferably citrate.

[0093] The method according to the invention may also comprise a step of demineralization and decalcification of the biological sample before bringing the biological sample into contact with the transparency composition and before the depigmentation step when a depigmentation step is necessary. A person skilled in the art can easily determine when a step of demineralization of the biological sample is necessary.

[0094] Preferably, the step of demineralization of the biological sample is carried out on hard tissues such as, for example, calcified tissues, bones, teeth, insects, fish, crustaceans, etc.

[0095] Preferably, the step of demineralizing the biological sample is carried out by bringing the biological sample into contact with a demineralization solution. The demineralization solution according to the invention preferably comprises at least one bivalent ion chelator. Preferably, the bivalent ion chelator is selected from the group consisting of EDTA (ethylenediaminetetraacetic acid), BAPTA (2, 2', 2", 2" '-[ethane-1,2-diylbis(oxy-2,i-phenylenenitrilo)]tetraacetic acid) and citrate.

[0096] Preferably, the step of demineralizing the biological sample is carried out at a temperature between 35 and 45°C, more preferably at approximately 42°C.

[0097] Preferably, the step of demineralizing the biological sample is carried out for a duration that depends on the type of biological sample used and its size. Preferably, the step of demineralizing the biological sample is carried out for a duration of between 10 minutes and a few days. The demineralization duration adapted to the biological sample used can be easily determined by a person skilled in the art.

[0098] Preferably, the method according to the invention further comprises a step of stopping the transparency. The step of stopping the transparency is preferably carried out by immersing the biological sample in a solution of refractive index homogenizing agent. Preferably, the step of stopping the transparency is carried out by immersing the biological sample in a solution of refractive index homogenizing agent after removal of the delipidation and permeabilization agent.

[0099] Preferably, the transparency stopping step is carried out by immersing the biological sample in TDE. More preferably, the transparency stopping step is carried out by immersing the biological sample in TDE at a concentration of 60%. Imaging may subsequently take place in the same buffer.

[0100] Preferably, the biological sample can be stored for several months in the refractive index homogenizing agent solution without delipidation and permeabilization agent. Preferably, the biological sample can be stored for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months in the refractive index homogenizing agent solution without delipidation agent.

[0101] Preferably, the transparency method according to the invention is reversible, that is to say that the biological sample made transparent by the method according to the invention becomes opaque again when it is immersed in a buffer. Preferably, at least 50% of the biological sample, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% of the biological sample regains its initial opacity when it is immersed in a buffer solution, such as for example a PBS solution, a PIPES solution, a HEPES solution, a MOPS solution, and a Tris-HCl solution.

[0102] According to a particular embodiment, the method according to the invention comprises: A step of depigmenting a biological sample by bringing the biological sample according to the invention into contact with a pretreatment composition according to the invention comprising H202, an alcohol, in particular ethanol, and citrate;

[0103] A transparency step by bringing the depigmented biological sample into contact with the transparency composition according to the invention;

[0104] A step of stopping transparency by immersing the biological sample in a TDE solution, preferably 60% TDE.

[0105] According to a particular embodiment, the method further comprises, before the depigmentation step, a step of decalcification of the biological sample.

[0106] According to one embodiment of the invention, the method according to the invention further comprises one or more detection and / or characterization steps such as:

[0107] (i) Detection of autofluorescence or other intrinsic contrasts, such as second harmonic (SHG) or third harmonic (TH G);

[0108] (ii) Staining the biological sample using one or more fluorescent dyes;

[0109] (iii) Immunofluorescence;

[0110] (iv) Optical phase or density imaging;

[0111] (v) Immunohistochemistry or fluorescent histochemistry

[0112] (vi) Expression of fluorescent protein produced by transgenic animals e.g. mice, fish like zebrafish, insects, (like tdTomato, AmCyan, GFP...).

[0113] According to a particular embodiment, the method according to the invention comprises a step of marking a target of the biological sample with a dye or a marking agent, preferably coupled to a fluorescent molecule. Preferably, the step of marking the biological sample is carried out before the step of bringing the biological sample into contact with the transparency composition according to the invention.

[0114] Preferably, the labeling step makes it possible to highlight and / or quantify targets of the biological sample.

[0115] According to a particular embodiment, the method according to the invention comprises a direct or indirect immunolabeling step.

[0116] The labeling or immunolabeling step can be carried out according to conventional labeling techniques well known to those skilled in the art. For example, it is possible to use antibodies, fluorescent probes, proteins, proteins produced by genetically modified organisms, etc., for labeling a target of the biological sample.

[0117] Examples of targets of the biological sample include biomolecules or synthetic molecules of the biological sample. Preferably, the targets of the biological sample are selected from the group consisting of specific cellular and tissue constituents, such as nucleic acids, antigens, proteins, peptides, fragments containing amino acids, oligonucleotides, including oligonucleotides containing DNA bases, RNA bases, DNA and RNA bases, and synthetic bases, nucleic acid fragments, lipids, hormones, vitamins, and sugars of the biological sample.

[0118] Examples of species that can be used to label a target of the biological sample include antibodies or an antibody fragment such as a monoclonal antibody, a polyclonal antibody, an immunoglobulin G (IgG), an immunoglobulin M (IgM), a single-chain variable fragment (scFv) antibody, a nanoantibody, an antigen-binding fragment (Fab), etc.

[0119] Preferably, the fluorescent molecules according to the invention, also known as fluorochrome or fluorophore, are selected from the group consisting of coumarins such as hydroxycoumarin, methoxycoumarin, aminocoumarin, cyanines, such as Cy5, Cy2, Cy3, and Cy7, a xanthene-based dye such as fluorescein and rhodamine in particular FAM, HEX, and TRITC, the Alexa Fluor range in particular Alexa Fluor 488, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, and Alexa Fluor 660, tamara, Red6i3, and Texas red.

[0120] The biological sample according to the invention can be observed by optical imaging. Preferably, the transparency obtained using the transparency composition according to the invention makes it possible to improve the observation capacity and the signal detection sensitivity of fluorescent and non-fluorescent structures.

[0121] Any optical imaging technique well known to those skilled in the art may be used to observe the biological sample according to the invention. Preferably, the optical imaging techniques used in the method according to the invention include intensity, phase, lifetime imaging, light sheet microscopy, confocal microscopy, fluorescence microscopy, conventional transmitted light microscopy, and two-photon excitation microscopy, dissection microscopy, fluorescence plaque detection, nanoscopy, spectroscopy and optical tomography.

[0122] Images obtained by optical imaging techniques can be processed with standard 3D reconstruction software well known to those skilled in the art.

[0123] According to a particular embodiment, the method according to the invention comprises:

[0124] A step of depigmenting a biological sample by bringing the biological sample according to the invention into contact with a pretreatment composition according to the invention;

[0125] A step of marking a target of the biological sample with a marking agent; A step of making transparent by bringing the biological sample into contact with the transparentizing composition according to the invention;

[0126] A step of stopping transparency by immersing the biological sample in a TDE solution, preferably 60% TDE.

[0127] According to a particular embodiment of the invention, a method according to the invention comprising the immunolabeling of the biological sample may comprise the following steps:

[0128] - A step of dehydration of the biological sample;

[0129] - A step of permeabilization of the biological sample;

[0130] - A step of blocking non-specific sites

[0131] - A labeling step with a primary antibody specific to the target, the incubation time depending on the thickness of the biological sample and being easily determined by those skilled in the art;

[0132] - A step of washing off excess antibodies;

[0133] - A labeling step with a secondary antibody which recognizes the primary antibody and carries the fluorescent molecule;

[0134] - A step of washing off excess antibodies;

[0135] - A transparency step by bringing the biological sample into contact with the transparency composition according to the invention;

[0136] - A step to stop transparency by immersing the biological sample in a TDE solution, preferably 60% TDE.

[0137] Alternatively, a method according to the invention comprising immunolabeling the biological sample may comprise the following steps:

[0138] - A transparency step by bringing the biological sample into contact with the transparency composition according to the invention;

[0139] - A step of stopping the transparency by immersing the biological sample in a TDE solution, preferably 60% TDE intended to evaluate the interest of the sample by initial imaging;

[0140] - An extensive washing step to remove the transparency medium and rehydrate the sample;

[0141] - A step of blocking non-specific sites;

[0142] - A labeling step with a primary antibody specific to the target, the incubation time depends on the thickness of the biological sample and can be easily determined by those skilled in the art;

[0143] - A step of washing off excess antibodies;

[0144] - A labeling step with a secondary antibody that recognizes the primary antibody and carries the fluorescent molecule; - A step of washing off excess antibody;

[0145] - A step to stop transparency by immersing the biological sample in a TDE solution, preferably 60% TDE.

[0146] Kit

[0147] Preferably, the kit according to the invention comprises a container comprising a composition comprising a delipidation and permeabilization agent according to the invention.

[0148] Preferably, the kit according to the invention comprises a second container comprising a composition comprising a refractive index homogenizing agent, such as thiodiglycol (TDE).

[0149] Preferably, the container comprises a composition comprising a delipidation and permeabilization agent according to the invention, in particular a terpene, and also comprises a non-ionic surfactant according to the invention.

[0150] Preferably, the container comprising a composition comprising a refractive index homogenizing agent, such as thiodiglycol (TDE) also comprises a fluorescence alteration retarding agent also known as an antifading agent.

[0151] The kit according to the present invention may further comprise a depigmentation composition according to the invention comprising H202, an alcohol, such as ethanol and a chelating agent, such as citrate.

[0152] The kit according to the present invention may further comprise a demineralization solution preferably comprising a divalent ion chelator, such as EDTA, BAPTA or citrate.

[0153] The kit according to the present invention may further comprise instructions for use of the kit and the clearing composition.

[0154]

[0155] Figure 1 represents a curve representing the percentage of contrast (%) measured after photography of the samples tested during transparency as a function of time (minutes). The samples are 1 mm slices of 12-month-old mouse brains transparentized in a transparency composition according to the invention and imaged in a 60% TDE composition.

[0156] Figure 2 represents a spider before carrying out the transparency process according to the invention.

[0157] Figure 3 shows a spider after undergoing a 2-day depigmentation step.

[0158] Figure 4:

[0159] Figure 4 represents a spider after having undergone a transparency step according to the invention of 40I1.

[0160] Example 1

[0161] The inventors evaluated the transparency time of biological samples with the transparency composition according to the invention and with TDE alone.

[0162] 1. Materials and methods

[0163] 1 mm thick samples of 12-month-old mouse brain and 1-month-old mouse brain were depigmented overnight in a composition comprising H2O2, ethanol, and citrate and buffer.

[0164] 2 different transparency compositions were tested:

[0165] (i) a composition; and

[0166] (ii) a composition comprising 60% TDE without D-limonene;

[0167] The 12-month-old and 1-month-old depigmented mouse brain samples were placed in a transparency composition according to the invention comprising D-limonene and 60% TDE in a transparent-bottomed tank placed on a target comprising black and white areas.

[0168] For comparison purposes, other 12-month-old and 1-month-old depigmented mouse brain samples were placed in a clearing composition comprising 60% TDE without D-limonene in a clear-bottomed tank placed on a target with black and white areas.

[0169] In order to evaluate the effect of temperature on the transparency rate, 1 mm thick samples of depigmented 12-month-old mouse brain were placed in a transparency composition according to the invention comprising D-limonene and 60% TDE at 37°C or in a composition comprising 60% TDE without D-limonene at 37°C in a transparent-bottomed tank placed on a target comprising black and white areas.

[0170] The level of transparency was assessed using the Michelson contrast measured after photographing the sample during transparency. The intensity of the white and black areas was determined using ImageJ software (Schneider et al. (2012) Nature Methods, 9: 671-675). The transparency of the samples using the transparency composition according to the invention is compared to that of those transparentized by TDE 60% alone. The data are analyzed by determining the Michelson contrast. The curve i / contrast = fct time (minutes) gives the T1 / 2 (time-lag) which corresponds to the time required to obtain 50% of the maximum contrast.

[0171] 2. Results

[0172] In Figure 1, it can be seen that the transparency obtained with the transparentizing composition according to the invention is more effective than with TDE.

[0173] The time required for transparency is shown in Table 1 below:

[0174] Table 1:

[0175] Maximum transparency for this fabric is achieved more quickly when TDE is in the presence of D-limonene.

[0176] The time required for transparency at a temperature of 37°C is shown in Table 2 below:

[0177] Table 2:

[0178] The speed of transparency was doubled by increasing the temperature at which the biological sample was brought into contact with the transparency composition. Example 2

[0179] The inventors tested the transparency method according to the invention on a spider (see Figure 2).

[0180] A whole spider underwent a first depigmentation step using a depigmentation composition according to the invention comprising H2O2, ethanol and citrate. The spider was completely depigmented (see Figure 3). The depigmented spider was then embedded in agarose and underwent a transparencing step by contacting with a transparencing composition comprising D-limonene and 60% TDE. The spider embedded in the agarose was completely transparencing (see Figure 4).

Claims

Claims 1. Composition for transparentizing biological samples, comprising at least: - a defatting and permeabilizing agent; - a refractive index homogenizing agent; in which the delipidation and permeabilization agent is a compound chosen from terpenes or terpenoids, preferably terpenes.

2. A transparizing composition according to claim 1, wherein the defatting and permeabilizing agent is the monoterpene limonene, preferably D-limonene.

3. Transparency composition according to claim 1 or 2, comprising from 0.5% to 10%, from 0.5% to 5%, from 0.5% to 2%, from 0.5% to 1.5%, advantageously 1%, by volume / volume of delipidation agent relative to the total volume of the transparency composition.

4. A transparizing composition according to any one of claims 1 to 3, further comprising a non-ionic surfactant.

5. A clearing composition according to claim 4, wherein the non-ionic surfactant is selected from the group consisting of triton X-100 and polysorbates such as polysorbate 80 (TWEEN80) or polysorbate 20 (TWEEN20), preferably the non-ionic surfactant is triton X-100.

6. A transparency composition according to any one of claims 1 to 5, wherein the refractive index homogenizing agent is selected from the group consisting of thiodiglycol (TDE), ethyl cinnamate and sucrose, preferably the refractive index homogenizing agent is TDE.

7. A transparency composition according to any one of claims 1 to 6, further comprising an agent delaying the alteration of fluorescence.

8. Transparency composition according to any one of claims 1 to 7, being an aqueous composition.

9. Use of a transparency composition as defined in any one of claims 1 to 8, for making a biological sample transparent.

10. A method for making a biological sample transparent comprising a step of bringing a biological sample into contact with a transparency composition as defined in any one of claims 1 to 8.

11. The method of claim 10, further comprising a step of immersing the biological sample in a solution of refractive index homogenizing agent, after removal of the delipidating agent, in order to stop the transparency of the biological sample.

12. Method according to claim 10 or 11, further comprising a step of depigmenting the biological sample before the step of contacting the biological sample with the transparency composition.

13. The method of claim 12, wherein the depigmentation is carried out by contacting the biological sample with a composition comprising at least H202, an alcohol, such as ethanol, and a chelating agent, such as citrates.

14. Method according to claim 12 or 13, further comprising a step of demineralizing the biological sample before the depigmentation step and / or before bringing the biological sample into contact with the transparency composition.

15. Method according to any one of claims 10 to 14, further comprising a step of labeling a biomolecule or a synthetic molecule of the biological sample with a fluorophore before the step of bringing the biological sample into contact with the transparency composition, the biomolecule or synthetic molecule being chosen from a nucleic acid, a protein, a hormone, a vitamin or a sugar.

16. Kit for making a biological sample transparent comprising: - a composition comprising a defatting and permeabilizing agent, such as D-limonene, - a composition comprising a refractive index homogenizing agent, such as thiodiglycol (TDE), - a depigmentation composition comprising H202, an alcohol and a chelating agent, such as citrates, - a demineralization solution comprising a divalent ion chelator, such as EDTA, BAPTA or citrate.

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