CN110694078A - Skull light transparent reagent suitable for visible-near infrared two regions - Google Patents
Skull light transparent reagent suitable for visible-near infrared two regions Download PDFInfo
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- CN110694078A CN110694078A CN201911003204.XA CN201911003204A CN110694078A CN 110694078 A CN110694078 A CN 110694078A CN 201911003204 A CN201911003204 A CN 201911003204A CN 110694078 A CN110694078 A CN 110694078A
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Abstract
The invention provides a skull light transparent reagent suitable for visible-near infrared two regions, which comprises a light transparent reagent and heavy water or DMSO-d used as a solvent6. The invention utilizes the characteristic that deuterium atom has one more neutron than hydrogen atom, so that the resonance energy level of heavy water (deuterium oxide) molecule is smaller than that of common water (hydrogen oxide) molecule, and the characteristic absorption peak is relatively red-shifted, thereby using the heavy water and DMSO-d6The tissue optical transparency reagent is prepared by replacing water and DMSO respectively, so that the transmittance of the optical transparency reagent in a near-infrared region can be effectively improved, and optical biological deep tissue imaging based on tissue optical transparency and the near-infrared region is realized.
Description
Technical Field
The invention relates to the technical field of biological imaging, in particular to a skull light transparent reagent suitable for a visible-near infrared region.
Background
The development of optical imaging technology has made it possible to observe biological tissue structures and vital movements with high resolution. However, the turbid nature of biological tissue limits the ability of light to propagate therethrough, severely limiting the use of optical imaging techniques for biological tissue imaging.
In the light transparent technology developed in recent years, chemical reagents are dripped on isolated tissues or living skin and skull, and the refractive index in biological tissues can be unified along with the infiltration of the reagents, so that the scattering is reduced, and the purpose of increasing the penetrating power of light in the tissues is achieved.
On the other hand, since the longer the wavelength of the light in a certain range, the smaller the scattering in the biological tissue, the light in the near infrared band (780-. At present, nonlinear optical imaging excited by a near-infrared two-region and fluorescence imaging excited by the near-infrared two-region both have better effects in the field of deep tissue imaging.
If an imaging means based on a near infrared band can be combined with a skull light transparent technology, the tissue imaging depth is expected to be further increased, and the observation of deep tissue structures and life activities is realized. However, current light transparent agents are developed primarily for the visible wavelength band, with the aim of reducing scattering of light by the tissue, while limiting absorption in addition to scattering, through which light penetrates in the tissue. In fact, the absorption of part of the near infrared band two by currently available light-transparent agents is very large, which limits the application of near infrared band-based imaging techniques in combination with light-transparency to deep tissue imaging.
The commonly used tissue light clearing agents at present comprise glycerol, sorbitol, sucrose, fructose, n-butanol, butanediol, propylene glycol, urea, sodium dodecyl benzene sulfonate, ethylene diamine tetraacetic acid and the like, and all the light clearing agents are dissolved by using water or dimethyl sulfoxide (DMSO) as a solvent for use (the DMSO itself is also a commonly used tissue light clearing agent). The inventor of the present application has found through research in the process of implementing the present invention that: water and dimethyl sulfoxide have obvious absorption in a near infrared two-region wave band, so that the transmittance of light with the wavelength in the absorption wave band in the optical transparency agent is influenced. Since imaging of a light-transparentized biological tissue is performed with the sample immersed in or coated with a light-transparentizing agent, the imaging efficiency is adversely affected when the excitation light or signal light of the absorption band is used in the imaging system. Disclosure of Invention
The invention provides a skull light transparent reagent suitable for a visible region and a near-infrared region, which increases the transmittance of the light transparent reagent to a near-infrared band on the basis of reducing biological tissue scattering.
Is suitable forVisible-near infrared two-region skull light transparent reagent, comprising light transparent reagent and heavy water or DMSO-d used as solvent6。
Further, the light transparent reagent is one or more of glycerol, sorbitol, sucrose, fructose, n-butanol, butanediol, propylene glycol, urea, sodium dodecyl benzene sulfonate and ethylene diamine tetraacetic acid.
Further, when the light transparent reagent is urea, the skull light transparent reagent is prepared by the following steps: dissolving ethanol and heavy water in a volume ratio of 3:1, preparing 75% ethanol solution, slowly dripping the solution on urea, continuously stirring, preparing saturated solution of urea, standing, and removing excessive urea precipitate.
Further, when the light transparent reagent is dodecylbenzene sulfonic acid, the configuration method of the skull light transparent reagent is as follows: preparing a sodium hydroxide heavy water solution with the molar concentration of 0.7M, and mixing the sodium hydroxide heavy water solution with dodecylbenzene sulfonic acid, wherein the pH value of the final solution is 7-8. .
The invention uses the heavy water and DMSO-d6The tissue optical transparency reagent is prepared by replacing water and DMSO respectively, so that the transmittance of the optical transparency reagent in a near-infrared region can be effectively improved, and optical biological deep tissue imaging based on tissue optical transparency and the near-infrared region is realized.
Drawings
FIG. 1(a) is the absorption spectra of water (dotted line) and heavy water (solid line) at the wavelength band of 350-2500 nm; FIG. 1(b) is DMSO (dotted line) and DMSO-d6(solid line) absorption spectrum at the 400-2000nm band, where the shaded portion represents the near infrared two-region band;
FIG. 2(a) is the absorption spectrum of a reagent I prepared with heavy water and a comparison reagent I prepared with water at the wavelength of 350-; FIG. 2(b) is the absorption spectrum of the second reagent prepared with heavy water and the second comparative reagent prepared with water at the wavelength of 350-;
FIG. 3 is a white light image of a mouse treated with a light-transparentizing agent formulated with water and a light-transparentizing agent formulated with heavy water using a stereomicroscope;
FIG. 4 is a graph of the triple frequency imaging signal intensity contrast for reagent one and reagent two in water and heavy water configurations, respectively, as imaging media;
FIG. 5 is a triple frequency imaging of cerebral blood vessels after the skull of a mouse is subjected to transparentization treatment.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings.
The invention provides that element 'hydrogen' in solvent molecules such as water and DMSO is replaced by element 'deuterium', and the transmittance of two solvents in a near-infrared region can be improved after the deuterium atom is replaced by the deuterium atom, namely the transmittance of an optical transparency agent in the near-infrared region can be improved on the premise of not changing the reduction effect of the optical transparency agent on biological tissue scattering.
Deuterium and hydrogen are isotopes of each other, have the same electron shell structure, but different atomic masses, and thus have the same chemical properties and different physical properties. Specifically, in the absorption spectrum line, because deuterium atoms have one more neutron than hydrogen atoms, the heavy water (deuterium oxide) molecules have smaller vibrational energy levels than ordinary water (hydrogen oxide) molecules, so that the characteristic absorption peak is relatively red-shifted, and the absorption intensity is weakened, as shown in fig. 1(a), therefore, the tissue optical transparency agent configured based on the heavy water can enable the absorption peak in the near-infrared two-zone to red-shift to be outside the near-infrared two-zone on the basis of not changing the biochemical characteristics of the original optical transparency agent, and the purpose of enhancing the transmittance in the near-infrared two-zone is achieved; similarly, DMSO-d is used6The same effect can be achieved by organizing light transparency instead of DMSO configuration, as shown in fig. 1 (b).
Examples of the applications
The invention selects a published living skull light transparency method which comprises two tissue light transparencies, wherein one tissue light transparence is saturated alcohol (75%) solution of urea, and the other tissue light transparence is high-concentration sodium dodecyl benzene sulfonate.
In one embodiment of the invention, heavy water is uniformly used for replacing water to prepare the reagent, and the specific preparation method comprises the following steps:
a first reagent: dissolving ethanol and heavy water in a volume ratio of 3:1, preparing a 75% ethanol solution, slowly dripping the solution on urea, continuously stirring, standing for 15 minutes, and removing redundant urea precipitate, wherein the mass-volume ratio of urea to 75% ethanol in the saturated solution is about 10: 3;
and a second reagent: preparing a 0.7M sodium hydroxide heavy water solution, mixing the sodium hydroxide heavy water solution with dodecylbenzene sulfonic acid, wherein the volume-mass ratio of the sodium hydroxide heavy water solution to the dodecylbenzene sulfonic acid is about 24:5, and the pH value of the final solution is 7-8.
Meanwhile, a first contrast agent and a second contrast agent which take water as a solvent are prepared as a contrast according to the same method.
When the light transparent method is used, firstly, the skin of the head of the mouse is cut off, the mucosa on the surface of the skull is erased, and the surface of the skull is dried; fixing the fixing piece with the hole on the surface of the blow-dried skull; dripping a first reagent into the hole of the fixing sheet fixed on the surface of the skull, and wiping the first reagent after 10-15 min; and dripping a second reagent into the hole of the fixing sheet, realizing the light transparentization of the skull tissue after 5-8min, and imaging by using an optical microscopic imaging system under the condition that the second reagent covers the surface of the skull.
First, the transmittances of the first and second contrast agents prepared with water and the first and second reagents prepared with heavy water in the visible-near infrared band were measured, respectively, and it was found that the transmittances of the two reagents prepared with heavy water in the visible light band were similar to those of the two reagents prepared with water, but were significantly higher in the near infrared band, as shown in fig. 2(a) and 2 (b).
Then, the mice were subjected to light-transparentizing treatment with a light-transparentizing agent prepared from water and a light-transparentizing agent prepared from heavy water, respectively, and white light imaging was performed with a body microscope, and the light-transparentizing effects were found to be the same, as shown in fig. 3:
in fig. 3 (a) is white light imaging of the skull surface; (b) is the intensity distribution of the dotted line in (a); (c) white light imaging of blood vessels under the skull is carried out after treatment by a visible-near infrared two-region skull light transparent reagent; (d) is the intensity distribution of the dotted line in (c); (e) the white light imaging of the skull surface is carried out by utilizing the recovered physiological saline; (f) is the intensity distribution of the dotted line in (e); (g) white light imaging of blood vessels under the skull is carried out after treatment by a visible-near infrared two-region skull light transparent reagent; (h) is the intensity distribution of the dotted line in (g); (i) is a partial magnified view of the content of the blue box in (c); (j) is the intensity distribution of the dashed line in (i); (k) is a partial magnified view of the content of the blue box in (g); (l) Is the intensity distribution of the dotted line in (k).
From this result, it was found that the effect of the light-transparentizing agent on suppressing tissue scattering, that is, the effect of transparentizing the tissue in the visible light band, is not reduced by preparing the light-transparentizing agent from heavy water.
Finally, imaging experiments were performed on the light transparency method before and after the modification using a set of scanning microscopy system carrying a femtosecond laser with a wavelength of 1560 nm:
a nanoprobe is selected, which can generate triple frequency of 520nm wavelength under 1560nm excitation. The dispersion of the probe was placed in a capillary glass tube, and frequency tripled imaging was performed using the above microscope system, and the media between the objective lens (25 ×, working distance 2mm) and the capillary glass tube were two reagents prepared with heavy water and two reagents prepared with water, respectively.
As shown in fig. 4, experiments have shown that when reagent one is formulated with heavy water as the imaging medium, the signal intensity obtained is much higher than when reagent one is formulated with water as the imaging medium; when reagent two, formulated with heavy water, was used as the imaging medium, the signal intensity obtained was much higher than when reagent two, formulated with water, was used as the imaging medium. Experiments prove that the light transparent reagent prepared by heavy water has better performance in optical imaging based on a near infrared two-region.
The reagent I and the reagent II which are prepared by using the heavy water are used for the skull penetrating imaging of the near infrared two-region excitation after the skull is optically transparent: the nanoprobe is injected into the circulatory system of a mouse through the tail vein, and the skull of the mouse is subjected to transparentization treatment by the light transparentization method and then subjected to frequency tripling cerebrovascular imaging, so that the imaging depth of 650 mu m is realized (as shown in figure 5). The application capability of the invention in the optical biological imaging based on the near infrared two-region is verified from the living body level.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (4)
1. A skull light transparent reagent suitable for visible-near infrared regions is characterized in that: comprises a light transparent reagent and heavy water or DMSO-d used as a solvent6。
2. The cranial light clearing agent suitable for the visible-near infrared region of claim 1, wherein: the light transparent reagent is one or more of glycerol, sorbitol, sucrose, fructose, n-butanol, butanediol, propylene glycol, urea, sodium dodecyl benzene sulfonate and ethylene diamine tetraacetic acid.
3. The skull light-transparent reagent suitable for the visible-near infrared region as defined in claim 2, wherein: when the light transparent reagent is urea, the skull light transparent reagent is prepared by the following steps: dissolving ethanol and heavy water in a volume ratio of 3:1, preparing 75% ethanol solution, slowly dripping the solution on urea, continuously stirring, preparing saturated solution of urea, standing, and removing excessive urea precipitate.
4. The skull light-transparent reagent suitable for the visible-near infrared region as defined in claim 2, wherein: when the light transparent reagent is dodecylbenzene sulfonic acid, the configuration method of the skull light transparent reagent comprises the following steps: preparing a sodium hydroxide heavy water solution with the molar concentration of 0.7M, and mixing the sodium hydroxide heavy water solution with dodecylbenzene sulfonic acid, wherein the pH value of the final solution is 7-8.
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Cited By (4)
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| CN113171457A (en) * | 2021-04-21 | 2021-07-27 | 中国人民大学 | Adjuvant for improving near-infrared optical treatment effect and treatment method thereof |
| CN113324821A (en) * | 2021-06-01 | 2021-08-31 | 华中科技大学苏州脑空间信息研究院 | Method for quickly staining biological sample and acquiring three-dimensional data |
| CN114288424A (en) * | 2021-12-22 | 2022-04-08 | 湛江中心人民医院 | Tissue clearing maintenance reagent and kit for tissue clearing |
| CN115554421A (en) * | 2022-04-26 | 2023-01-03 | 湛江中心人民医院 | Skin tissue light transparent reagent and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113324821A (en) * | 2021-06-01 | 2021-08-31 | 华中科技大学苏州脑空间信息研究院 | Method for quickly staining biological sample and acquiring three-dimensional data |
| CN114288424A (en) * | 2021-12-22 | 2022-04-08 | 湛江中心人民医院 | Tissue clearing maintenance reagent and kit for tissue clearing |
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| CN115554421A (en) * | 2022-04-26 | 2023-01-03 | 湛江中心人民医院 | Skin tissue light transparent reagent and preparation method and application thereof |
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