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Method for rendering tissue transparent, reagent for rendering tissue transparent, and tissue observation method

Foreign code F170009173
File No. AF13-04WO
Posted date Sep 12, 2017
Country EPO
Application number 13872734
Gazette No. 2950077
Date of filing Nov 20, 2013
Gazette Date Dec 2, 2015
International application number JP2013006811
International publication number WO2014115206
Date of international filing Nov 20, 2013
Date of international publication Jul 31, 2014
Priority data
  • P2013-012889 (Jan 28, 2013) JP
  • 2013JP04369 (Jul 17, 2013) WO
  • 2013JP06811 (Nov 20, 2013) WO
Title Method for rendering tissue transparent, reagent for rendering tissue transparent, and tissue observation method
Abstract A method for rendering tissue transparent, comprising a procedure of immersing the tissue in a water-soluble solvent comprising 2,2'-thiodiethanol and at least one of glycerol and a nonionic organoiodine compound is provided as a technique capable of sufficiently rendering various organs transparent without causing their changes by a simple operation without using any poisonous or dangerous organic solvent.In the method for rendering tissue transparent, a mixed solvent of 2,2'-thiodiethanol, glycerol, and a nonionic organoiodine compound aqueous solution is preferably used as the water-soluble solvent.
Outline of related art and contending technology Background Art
With the recent progress of genetic transformation and gene introduction technologies, it has been carried out to label only specific cells in any of various organs with a fluorescent protein for observation.For example, attempts have been made to selectively label various neural circuits in the brain with fluorescence to 3-dimensionally visualize the neural circuits using the fluorescence as an indicator for reconstruction.
Conventionally, the observation of the internal tissue of an organ has been performed by mechanically slicing the fixed and embedded organ (or organ piece) to prepare tissue sections and observing each section under a light microscope.In recent years, the observed image of the internal tissue of an organ has come to be obtained by optically "cutting" the organ using a confocal laser microscope or a multiphoton excitation fluorescence microscope.
When a tissue structure, such as a neural circuit, is 3-dimensionally observed, the above mechanical method requires the preparation of many continuous tissue sections and the piling-up of their fluorescence images, which is accompanied by great labor.In contrast, the optical method has the problem of making the acquisition of fluorescence images more difficult (observation depth limit) as an increase in the depth of an observed site from the organ surface because light scatters inside the organ.
The observation depth limit is generally considered to be on the order of 0.15 mm for the confocal laser microscope and on the order of up to 4 mm for the two-photon excitation fluorescence microscope.For example, for the brain of mice widely used for research, since the cortex as the outer layer has a thickness of about 1 mm, it is necessary to expand the observation depth limit to several millimeters in order to observe the hippocampus or thalamus located on the more internal side of the brain than that for the cortex.
Accordingly, a technique for rendering an organ transparent has been studied as a technique for expanding the observation depth limit by suppressing the scattering of light inside the organ.For example, Non Patent Literature 1 reports that the brain/spinal cord was rendered transparent by a method using tetrahydrofuran.In addition, Patent Literature 1 and Non Patent Literature 2 state that the brain was successfully rendered transparent by a method using urea at a high concentration (Scale method).
Non Patent Literature 4 states that tissues, such as the thoracic ganglion of a dragonfly and the skin of a squid, were each successfully rendered transparent to a thickness of on the order of 0.6 mm using 2,2'-thiodiethanol.Non Patent Literature 5 preceding this literature states that 2,2'-thiodiethanol is used as a mounting medium.
Citation List
Patent Literature
Patent Literature 1: National Publication of International Patent Application No. 2013-522590 Non Patent Literature
Non Patent Literature 1: "Three-dimensional imaging of the unsectioned adult spinal cord to assess axon regeneration and glial responses after injury." Nature Medicine, 2011, Vol.18, No.1, pp.166-71
Non Patent Literature 2: "Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain." Nature Neuroscience, 2011, Vol.14, No.11, pp.1481-1488
Non Patent Literature 3: "Visual properties of transgenic rats harboring the channelrhodopsin-2 gene regulated by the thy-1.2 promoter." PLoS ONE, 2009, Vol.4, No.11, e7679
Non Patent Literature 4: "Labeling and confocal imaging of neurons in thick invertebrate tissue samples." Cold Spring Harbor Protocol, 2013
Non Patent Literature 5: "2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy." Microscopy Research and Technique, 2007, 70:1-9

Scope of claims [claim1]
1. A method for rendering tissue transparent, comprising a procedure of immersing the tissue in a water-soluble solvent comprising 2,2'-thiodiethanol and at least one of glycerol and a nonionic organoiodine compound.
[claim2]
2. The method for rendering tissue transparent according to claim 1, wherein the solvent is a mixed solution of 2,2'-thiodiethanol, glycerol, and a nonionic organoiodine compound aqueous solution wherein proportions by volume of 2,2'-thiodiethanol, glycerol, and a nonionic organoiodine compound aqueous solution having an iodine content of 40% are 10 to 50%, 1 to 20%, and 10 to 70%, respectively.
[claim3]
3. The method for rendering tissue transparent according to claim 1, wherein the solvent is a mixed solution of 2,2'-thiodiethanol and a nonionic organoiodine compound aqueous solution wherein proportions by volume of 2,2'-thiodiethanol and a nonionic organoiodine compound aqueous solution having an iodine content of 40% are 20 to 80% and 80 to 20%, respectively.
[claim4]
4. The method for rendering tissue transparent according to claim 1, wherein the solvent is a mixed solution of 2,2'-thiodiethanol and glycerol wherein proportions by volume of 2,2'-thiodiethanol and glycerol are 70 to 95% and 5 to 30%, respectively.
[claim5]
5. The method for rendering tissue transparent according to claim 1, wherein the solvent further comprises a sucrose aqueous solution.
[claim6]
6. The method for rendering tissue transparent according to any one of claims 1 to 5, wherein the solvent has a refractive index of 1.4 to 1.7.
[claim7]
7. The method for rendering tissue transparent according to any one of claims 1 to 6, wherein the tissue is one or more selected from the group consisting of the brain, spinal cord, liver, lung, heart, blood vessel, and cancer tissue.
[claim8]
8. The method for rendering tissue transparent according to claim 7, wherein the tissue has a thickness of 1 mm or more.
[claim9]
9. A reagent for rendering tissue transparent, comprising a water-soluble solvent comprising 2,2'-thiodiethanol and at least one of glycerol and a nonionic organoiodine compound.
[claim10]
10. A tissue observation method comprising a transparentization procedure which involves immersing tissue in a water-soluble solvent comprising 2,2'-thiodiethanol and at least one of glycerol and a nonionic organoiodine compound.
[claim11]
11. The tissue observation method according to claim 10, comprising
a labeling procedure involving fluorescently labeling the tissue,
the transparentization procedure involving immersing the fluorescently labeled tissue in the solvent, and
a detection procedure involving detecting fluorescence emitted from the fluorescent label in the transparentized tissue.
[claim12]
12. The tissue observation method according to claim 10, comprising
the transparentization procedure involving immersing the tissue in the solvent,
a labeling procedure involving fluorescently labeling the transparentized tissue, and
a detection procedure involving detecting fluorescence emitted from the fluorescent label in the transparentized and fluorescently labeled tissue.
[claim13]
13. The tissue observation method according to claim 11 or 12, wherein the fluorescence is observed using a fluorescence microscope, a fluorescence stereomicroscope, a confocal microscope, or a multiphoton microscope in the detection procedure.
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • ONODERA, Hiroshi
IPC(International Patent Classification)
Specified countries Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Reference ( R and D project ) CREST Creation of a Novel Technology towards Diagnosis and Treatment based on Understanding of Molecular Pathogenesis of Psychiatric and Neurological Disorders AREA
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