TW594005B - Aqueous tissue clearing solution - Google Patents

Abstract

This invention provides an aqueous tissue clearing solution whose composition is to clear a biological tissue to make it transparent. The clearing solution composition is prepared by adding one or many compounds of dimethyl sulfoxide, diatrizoate acid, ethylenediaminetetraacetic acid, glucamine, beta-nicotinamide adenine dinucleotide phosphate, sodium diatrizoate and polyoxyalkalene derivative into a suitable aqueous carrier. This invention utilizes aqueous clearing solution to make the biological tissue completely transparent so that its internal structure can be observed by a microscope.

Description

594005

Field of the Invention: The present invention relates to a solution for biological tissue analysis, and more particularly to a composition of a water-soluble (acuieous) clearing solution (clearing solution) that can make biological tissue transparent. Background of the Invention: In order to observe the planar or three-dimensional microstructure of living or non-living organisms in life sciences fields that specialize in cell biology, neurobiology, molecular biology, physiology, immunology, signal organisms, and developmental organisms, In addition to having a good microscope, such as a conjugate focal laser scanning microscope, to obtain high-resolution images, special attention must also be paid to the preparation of samples, recording of microscopic images, and image processing in order to use optics to observe some divergences. Fluorescent samples and the cellular or tissue structure of the organism. Among them, when preparing the samples to be used, it is necessary to first confirm the integrity of the overall two-dimensional structure of the sample during processing, such as in the steps of fixing or mounting. In general, conventional transmitted light microscopy are widely used in most biological research laboratories to observe biological cell structures. Before the biological tissue is observed with a microscope, it is usually cut into thin slices and stained with a staining agent to view the internal structure of the stained biological tissue. In order to increase the transparency of the tissue, the stained samples must first pass through Preparation of dehydration and lipid extraction processes to clarify the sample, for example: after dehydration of alcohol (a 1 c〇h〇1), xylene is often used to clarify paraffin

594005

Case No. 90115921 1. Description of the invention (2) The section is made transparent, and as a result, the dyeing is performed + imaging. The internal characteristics of / can be clearly focused. However, the extraction of the bath agent needs more time. U 1 The processing time of Wen Xi, and often causes tomb jealousy 1 TU on the morphology. ———— jealous oysters and dlstortion, and .-R no „, ^ M ^ In the case, this phenomenon needs to be avoided. For example, it can be quickly judged by cryosectionmgs (cryosectionmgs). ) Usually unprepared, wooden door, + will be broken and fixed well, these organizations

Sections are usually directly embedded in a water-soluble embedding solution, mainly glycerol A, and

There is no need to go through dehydration and clarification process, α avoids destroying the fine morphology of the tissue. In addition, when observed under a fluorescent microscope, it will be caused by the dissolution of the fluorescent dye. The image / month clarity is 1 °. Moreover, the thickness of the reorganization during the biological tissue microscopic observation, scanning, and image reorganization is known. It can only reach 丨 00 ～ 200 microns (// m), the reason is that the tissue specimen is not clear and transparent. In view of this, the present invention first proposes an aqueous clearing solution so that

Under the condition of free state and dehydration, the biological tissue is effectively transparent. As shown in Figures A through C, they are images of a complete insect Dayue® with a thickness of 50 m under an anatomical microscope. Among them, the first picture A does not show that the insect brain slices show normal opacity in a physiological saline solution. The first picture B shows the insect brain showing a translucent appearance in a 80% glycerol salt mixture. The image 'and the image in the first C figure are those who immerse the insect brain in the clarified solution of the present invention to achieve complete transparency' in order to effectively overcome the aforementioned shortcomings.

Page 5 594005 Case No. 90115921 V. Description of the invention (3) Purpose and summary of the invention: The main purpose of the invention is to make the liquid through the dehydration and clarification process.

Another object of the present invention is solution treatment. Obtained in a mirror. Still another object of the present invention is detailed and morphologically transparent. The following images of biological tissues are used to obtain a clear solution of one or a group of compounds such as diamine tetraacid phosphate, so that it is easy to understand the effect below. Putting it in a suitable biological tissue completely by implementing the present invention aims to provide a water-soluble tissue to clarify and dissolve completely transparently, and it is not necessary to make the tissue transparent as is conventionally required. It is to provide a kind of clarification through water-soluble tissue, so that it has higher resolution in fluorescent and non-fluorescent optical microscopy. The water-soluble tissue clarification solution is used to make biological tissues without slicing and damaging the tissue. The water-soluble tissue clarification solution of the present invention is acer, diethylaminotriiodobenzoic acid, hypothionine, and yS-tobaccoamide. Sodium adenine dinucleoside iodobenzoate and polyoxyalkylene derivatives are used in water-soluble solutions to make use of this water-solubility. The example is accompanied by a detailed description of the attached drawings, and detailed descriptions of Danggen, technical valleys, features, and the accomplishments of the invention: The present invention relates to a water-soluble clarified solution that makes biological tissues transparent, and is used to increase biological Tissue transparency, the clear solution composition includes one or more dimethyl suifoxide

594005 90115921 V. Description of the invention (4) ethylenediaminetetraacetic acid, glucamine, yg-tobaccoamine adenine dinucleotide phosphate Γ · V p'nicotinamide adenine dinucleotide phosphate), two Derivatives of sodium aminodiatrizoate and polyoxyalkylenes (trademark Tween 20, used as emulsifiers and detergents) and place all of the above ingredients in a suitable To obtain a clear solution ', and the pH value of the clear solution is in the range of 5-10. Among them, the above-mentioned biological tissue system includes animal and plant cells, biological officials, biological compounds, and biological donations. Regardless of the use of the conventional fixation method, the biological tissue is directly immersed in the clarified solution of the present invention, and after a period of incubation, a biological tissue with better transparency (franSparenCy) can be formed. When a specific internal structure of interest is marked by an antibody fluorescent fluorescence or traditional dyes, the use of the solution of the foregoing components can produce better transparency, and by the goal of the calibration , So that the dyed structure can produce better images and better detection sensitivity (S en S 丨 ti V 丨 ty). Therefore, in optical detection methods, such as a confocal microscope (c ο nf occa 1 microscopy), fluorescence microscopy, traditional light microscopy, dissection microscope ( dissecting microscopy), flow 3: counter (fiow cytometry), spectrophotometer (spectrophotometry), fluorescent light plate detection

594005 Amendment-Case No. 90159921 5. In the application period of the method of the invention (5) (fluoreScence piate detecti0n) and fluorescence chip detection, the transparency formed by using the water-soluble clear solution of the present invention , Will improve the observation and signal detection capabilities of labor and non-fluorescent cell structures. Confocal microscopes provide removal of out-of-f0CUS background fluorescence noise from a thick-slab sample with a cursor, and record the X, Y, and Z coordinates of the target object so that it can be drawn. And analyze the three-dimensional image into three-dimensional space. Generally, biological tissues are thicker than 100 microns and about 5 to 10 cells thick, which usually hinders the efficiency of fluorescence excitation and detection and leads to signal attenuation. On the other hand, continuous alcohol dehydration and nail polish are generally used. Base salicylate-penetration clarification often results in diffusion of the calibrated fluorescein. In the clarification solution of the present invention, clarifying (c 1 earing) and embedding (embedding) the structure of the cursor will greatly overcome these problems, such as when the glycerol-salt embedded in 80% In the mixture, the neural olfactory globules in an insect antennae leaf (ante η na 1 1 〇be) can only be shared by

The light focus laser scanning microscope observed 100 micrometers below the surface, as in the second A f; in contrast, when this sample was clarified and embedded in the clarification / valley fluid of the present invention, it could still be clearly observed The internal structure is 200 microns deeper than the surface, as shown in the second figure B. Furthermore, some lipophilic fluorescent dyes used to calibrate the internal structure of biological tissues, such as the NBD-neuraminyl CMBD used in the second figure -ceram ide) coloring the structure of all cell membranes will gradually dissolve in the glycerol-salt mixture, but will not dissolve in the clear solution of the present invention. The following is a more specific embodiment to illustrate the present invention.

594005 Case No. 90115921 _η Name Amendment V. Description of the Invention (6) [Example 1] Microscopic image of thick sheet tissue marked with a fluorescent probe. A kind of insect brain of a cockroach (Diploptera punctata), whose thickness is greater than 500 micrometers, is used as an example for verification: First, the neuropil structure and the neuronal somat in the brain are related to each other. Grease cell membrane probe (probe) NBD C6-Neuramin and DNA probe prop idium iodide staining agent for fluorescent staining; appropriate fixation in 4% paraformaldehyde solution After that, the nuclei in the brain was digested with RNase at 50 g / W, and iodized with 20 vg / mi with physiological saline (ph0sphate buffere (i saline, PBS)). Propidium staining and calibration. Next, after a brief rinse with physiological saline, the cell membrane was stained with dipyridine to map 0.435mMiNBD Ce-neuraminamine; then the brain tissue was directly immersed in the clear solution of the present invention Make it completely transparent for one hour; in order to prevent the sample from being squeezed by the cover glass, the sample is placed in an annular ring with a height of about 600, m, and then the image is ordered by a conjugate focus laser scanning microscope, and the whole brain is The fluorescent structure can be directly under the traditional structure; Chagang or a micro-focus laser scanning microscope is used to form a microscopic image. The second picture shows the three-dimensional space containing all internal nerve fibers = brain microscope The image is formed by reorganizing the 168 common thunder // ΓΓΓΤ image. If the brain tissue text has sufficient transparency to provide the emperor, # f; and: 有: 敫 will cause the microscopic imaging to become blurred or impossible. Imaging. In addition to clarifying and calibrating the calibration tissue, the clarification solution of the present invention

Page 9 ', laser penetration and Xin 594005 Case No. 90115921 Fifth, invention description (7) The liquid can also be used in tissue sections such as frozen sections and vibration sections [Example 2] Utilization Microscopic image of thick sheet tissue marked with non-fluorescent dyes. Take the brain of Acheta domesticus as an example for verification. First, the brain was immersed in 4% paraformaldehyde physiological food rice / a + min® water and frozen for $ 2 hours, and then cooled with ice. The saline was washed three times for ten minutes each time. This brain tissue was immersed in a physiological saline solution containing 1% trinitrazine, τ · β lotr (Tri ton χ-100) for 16 hours at 4 QC. It was completely infiltrated. After being washed with physiological saline, the catalyst enzyme activity of NADPH-diaphorase was tested at 27 ° C. 1% trinitrotoluene, lmM′々 咄 ⑽ ^ and 0.5 mM NBT in 100 # 1 trihydrochloride (Tris_Hcl, 50 mM, pH = 7 4) for 2 hours; this chemical reaction will produce a blue precipitate (Β — formazan) at NADPH-diaPhorase, and then washed tissue with 72% trihydrochloride buffer solution containing 1% dichlorotoluene for 72 hours, ^ After 6, the entire brain was directly immersed in the clarification of the present invention Made in solution: One hour to make it clear and completely transparent; to avoid brain pressure, the sample The clear solution was placed in the same bucket Π3 e + · - the slide under a microscope Ο sectional Enough tissue _ ,,, in count% < affinity observed and photographed against light, or to Zeiss.

An Axiphot light microscope was used to perform f R, .u unsubscribe observations. The fourth picture shows the high fermentation method of NADPH-diaphorase. Master Pu: said each m. LL, ^ ', the birth is manifested in the mushroom-like body that produces Shen Dian, because this brain passes through Right working soil foot, month, and valley fluid are transparentized, so that the sturdy body of 1 Neiqiu can be clearly seen. Han, the early state of the inner pupa, the clarified solution of the present invention can also pupa,

Mil I --------- 乂 is applied to other non-fluorescent dyes. Page 10 594005 Case No. 90Π5 Loss 1 V. Description of the invention (8) Sheng Yi __Δ_ ^ A positive tissue slice, such as freezing Sectioning, vibration sectioning, etc. [Example 3] Single cell: image using fluorescent or non-fluorescent probe (probe). The single cells fixed on the glass slide (de) were directly clarified and embedded in the Qingluo solution. The water-solubility characteristics of this clarified solution: the direct treatment of antibodies with fluorescent staining or fluorescent dyes Like. The fifth figure shows the human fibrous tissue blast cells; microscopic images under a scanning microscope.

Date: The degree of fluorescence makes the fluorescence stimulation efficiency and fluorescence emission detective J's relatively improved. Therefore, a fluorescent light ca n’t be removed more effectively. The Huai'an ancient hall, Standing out seven, + 1 person and then ^ back to the sharpness of the image. This method can also be effectively used to observe spontaneous fluorescent tissues. Example 4: Drosophila mutants with green fluorescent protein or plant: weaving. As shown in the sixth figure: it is a three-dimensional microscopic reorganized image of pollen grains with a diameter of 100, and it is obtained with a depth interval of 5 // m. The shape of the surface is restructured by the yoke-focus optical cross section. The above-mentioned embodiment is only at the point of inflammation% ηη -I »a η, the purpose of which is to familiarize yourself with this: technical ideas and special features. According to the implementation, when it is impossible to explain the spirit of the present invention according to the present invention with ===, = range ', that is, a large β corpus / r TF average 4 change or modification library is covered by the patent scope of the present invention. Two cases No. 90115921 Schematic illustrations Schematic description:: Micro A microscope to U: is "" The frequency of the beetle brain in different processing solutions under anatomical solution. The brain tissue in the physiological mixture containing salt = like the figure obtained in the clarified solution of the glycerol salt invention of the younger brother = 525 microns. ^ U ~ image, the thickness of this brain is the second picture A is a continuous image of a microscopic image of an insect's antennal leaf, the second picture is clear and the mother is 50 micrometers apart ^ picture 13 *; about 100 micrometers only . The microscopic image of the clarification solution can be clarified and embedded in the structure of the present invention. π to / clothed inside 200 micrometers below the surface. The third picture is the recombination diagram of the common emperor's brain. Yoke ... Tian Shezhi describes the three-dimensional space of the microscopic image. The picture shows the 蟋蟀 brain passing through the clear solution to be observed under a transmissive optical microscope, in which ^ ′ is through-image. Micrograph of Ligu-shaped body The fifth image is a microscopic image of human fibrous tissue mother cells under a high resolution microscope. The laser scan of jin / yoke focus shows the sixth picture is the yoke of pollen grains with a diameter of 1000 microns.

Claims (1)

594005 --- Case No. 90115921 __ ^ VI. Application for patent scope 9 Use = The method described in item 8 of the patent scope, where the sample can be observed with a total of coke laser scanning microscopes. 10. The method according to item 8 of the Chinese Patent Application, wherein the sample can be observed with a common optical microscope. Π. The method described in item 8 of the scope of patent application, wherein the sample can be observed with a flow counter. 12. The method according to item 8 of the scope of patent application, wherein the sample can be directly observed with an eye or a dissecting microscope. 13. A method for observing biological tissues, comprising the following steps: first, processing biological tissues through a water-soluble tissue clarification solution to obtain a clear and transparent biological tissue sample, and the water-soluble tissue clarification solution is Include a suitable water-soluble carrier to add one or more of dimethyl sulfene, diethylaminotriiodobenzoic acid, ethinyldiaminetetraacetic acid, reduced glucose, amine, stone-tocopheryl adenine dinuclear Derivatives of uric acid phosphate, sodium diethylaminotriammonium benzate, and polyoxyalkylene; and observe the sample with a microscope to obtain an image of the internal structure of the sample. ° 14. The method according to item 13 of the patent application scope, wherein the microscope is a confocal laser scanning microscope. 15. The method according to item 13 of the scope of patent application, wherein the microscope is an ordinary optical microscope. Date of application: Category ϋη〇 (/ \) ^ (The above columns are filled by the Office) Amendment No. · 90115921 Amendment Notice This invention patent specification 594005 Chinese water-soluble tissue clarification solution Invention name English name (Chinese) 1. Name of Jiang Anshi inventor (English) 1. Nationality 1. Republic of China,. 丨, residence, residence 1. 101, Section 2, Guangfu Road, Hsinchu City ( Name) (Chinese) 1. Jiang Anshi's name (Name) (English) 1. Nationality 1. Republic of China applicant's residence, domicile (office) 1. Name of representative (No. 101, Section 2, Guangfu Road, Hsinchu City) 1. Name of the representative (English) 1. Page 594005 Case No. 90115921 Amendment ff ^ Amendment VI. Patent Application Scope Supplement 1 · A water-soluble tissue clarification solution, which is used to increase the transparency of biological tissues, the clarification The composition of the solution includes one or more of dimethylmethylenesulfonate, diethylaminotriiodobenzoic acid, hypoethyldiaminetetraacetic acid, reduced glucosamine, -tocobylamine adenine dinucleotide phosphate, Derivatives of sodium diethylaminotriiodobutyrate and polyoxyalkylenes are placed in a suitable water-soluble carrier. 2. The biological tissue line in the water-soluble tissue clarification solution described in item 1 of the patent application scope includes animal and plant cells. 3. The biological tissue system in the water-soluble tissue clarification solution as described in item 1 of the patent application scope includes biological organs. 4. The biological tissue system in the water-soluble tissue clarification solution described in item 1 of the patent application scope includes biological compounds and biological donations. 5. The pH value of the clarified solution composition in the water-soluble tissue clarified solution described in item 1 of the patent application range is between 5 and 10. 6. The water-soluble tissue clarification solution as described in item 1 of the scope of the patent application, further comprising an excess of sodium diethylaminotriiodobenzoate and diethylaminotriiodocarbamate. 7 · The water-soluble tissue clarification solution as described in item 1 of the patent scope of Shenyin, which further contains an excessive amount of dimethyl sulfene. 8 · A method for increasing the transparency of biological tissues, which is a step without dehydration treatment of organic solvents. The method is to process biological tissues through a water-soluble tissue clarification solution to obtain a clear and transparent biological tissue-like material. Other Page 13