Patent Application
20120081518
This application claims the benefit of TAIWAN Patent Application Ser. No. 099133619, filed on Oct. 1, 2010, which are herein incorporated by reference.
This invention relates generally to a method of visualizing a 3-dimensional structure of a thick biological tissue, and more particularly to a method of combining the optical-clearing process (use of immersion solution to reduce random scattering as light travels across media), confocal microscopy, and controlling tissue removal to visualize the 3-dimensional microstructure of a tissue block such as the endoscopic biopsy.
Three-dimensional (3D) microscopic visualization of tissue of interest is crucial in biomedical research. For example, 3D visualization of a suspicious area of the patient's biopsy provides a global view of the tissue organization. This feature is particularly valuable when intricate vascular and neural networks are the prime targets for inspection. Although the microtome-based 2-dimensional (2D) histology is the current standard method for tissue analysis, diagnostic errors and inconclusive results are embedded in the process due to incomplete sampling—only a few sections of the specimen are examined to represent the bulk tissue. Alternatively, biological tissues can be treated with an optical-clearing solution, such as the disclosed FocusClear solution (U.S. Pat. No. 6,472,216; Taiwan R.O.C patent 206390) for deep-tissue microscopy. The optical-clearing process is to immerse the tissue in the optical-clearing solution, which has a high refractive index, to reduce light scattering. For example, the application of the FocusClear solution (refractive index: 1.46) reduces the amount of refractive mismatch between tissue constituents (high refractive index, ˜1.46) and fluids (low refractive index; water has a refractive index at 1.33), therefore avoiding random scattering and making biological tissues transparent to facilitate laser penetration and fluorescence detection. Particularly, optical clearing is compatible with optical sectioning microscopy, such as confocal microscopy, which acquires optical slices from thick tissue specimens by excluding the out-of-focus signals from the focal plane, allowing 3D reconstruction of the acquired image stack. Combining the optical-clearing method and optical sectioning microscopy can significantly increase the imaging depth.
However, because optical clearing only reduces scattering, the limitation of the imaging depth is relaxed but still exists at the range ˜300 μm. For animal tissue of large size, such as human or mouse, this imaging depth cannot provide the field (or depth) for 3D characterization of the structure of interest at the centimeter level. Thus, it is crucial to devise an add-on to the disclosed FocusClear patent to extend the current image acquisition method.
Accordingly, there is still a need for a solution to enhance the imaging depth of optical-cleared biological tissues.
The present invention discloses a method for 3-dimensional microscopic visualization of thick biological tissues. This method includes: providing a thick biological tissue which is processed by an aqueous optical-clearing solution so as to allow light to travel across the thick biological tissue, providing a removal apparatus (or a cutter) to remove a portion of the thick biological tissue, using an image capturing apparatus to capture the image of a surface of the thick biological tissue, increasing a focal depth of the image capturing apparatus incrementally so as to capture the image stack from surface of the thick biological tissue to the boundary plane which is defined as the limitation of the imaging depth (the limitation is defined by the user according to required imaging quality of the user, because biological tissues scatter light, the resolution of the acquired image will be too low to satisfy user's demand of image quality when the focal path moves beyond the depth limitation), defining a removal plane above the boundary plane, executing a tissue removal to remove the thick biological tissue above removal plane which is the scattering source by using the removal apparatus, and increasing the focal depth to capture images of the thick biological tissue deeper than the boundary plane.
In one embodiment, the image capturing apparatus includes confocal microscopy, multi-photon laser scanning microscopy, and other optical sectioning microscopy. The optical-clearing solution includes FocusClear which was disclosed in U.S. Pat. No. 6,472,216. The thick biological tissue includes animal tissues, plant tissues, artificial tissues, or biomaterial tissues. The removal apparatus includes mechanical slicer, laser cutter, chemical erosion, or cauterization.
The removal plane is defined upper than the boundary plane or closer to the objective lens than the boundary plane (i.e. the depth of the removal plane is less than the depth of the boundary plane). The boundary plane is defined by the user according to the acquired image quality. The depth between the removal plane and the surface is less than the depth between the boundary plane and the surface. The distance between the boundary plane and the removal plane should be judged by user so as to prevent tissue under the boundary plane from distortion enforced by the tissue removal.
A plurality of tissue removal rounds is executed and each round follows the principal that the depth of the removal plane is less than the depth of boundary plane. This method acquires an image stack to provide the information of tissue's 3-dimensional microstructure with minimal interference from tissue sectioning as confronted by the conventional histological method.
The above objects, and other features and advantages of the present invention will become more apparent after reading the following detailed description when taken in conjunction with the drawings, in which:
a illustrates the shallow portion of the thick biological tissue is scanned incrementally from the surface of the thick biological tissue to the boundary plane by an image acquisition device such as a confocal microscope. Then, a removal plane is defined. The first image stack of the thick biological tissue is acquired after the scanning process.
b illustrates the first round of tissue removal of the thick biological tissue is executed and a new shallow portion is defined following the illustration in
The present invention will be described in detail using the following embodiments and it will be recognized that those descriptions and examples of embodiments are used to illustrate but not to limit the claims of the present invention. Hence, other than the embodiments described in the following, the present invention may be applied to the other substantially equivalent embodiments.
A method for visualizing the 3-dimensional microstructure of a thick biological tissue is disclosed in present invention. This method includes: utilizing a technique to increase the imaging depth of a tissue, scanning and capturing images of the tissue within the boundary plane where the limitation of imaging depth occurs, defining a removal plane upper than the boundary plane, and removing the tissue above the removal plane. Because the source of light scattering is largely reduced by tissue removal, the second round of imaging acquisition can be performed to extend the imaging depth to new boundary plane. Furthermore, removal of tissue upper than the boundary plane prevents the distortions caused by removal of tissue from interfering next round of imaging acquisition. Executing several rounds of the foregoing method, the thickness of biological tissues being visualized can be systematically increased, thus overcoming the depth limitation imposed by the conventional imaging approach.
The present invention discloses a method for observing the 3-dimensional microstructure of a thick biological tissue. In one embodiment, a thick biological tissue labeled by a fluorescent material is embedded and fixed in a gel. After immersing the opaque thick biological tissue in an optical-clearing solution, such as FocusClear (U.S. Pat. No. 6,472,216), the thick biological tissue 10 with thickness D (ex. about 600 μm) is placed on a scanning stage 20, as shown as in FIG. l. In one embodiment, the thick biological tissue includes animal tissues, plant tissue, artificial tissues, and biomaterial tissues. The technique of FocusClear was disclosed in U.S. Pat. No. 6,472,216, so the detail theory of FocusClear will not be described in the present invention. An image capturing apparatus 40, such as confocal microscopy, or multi-photon laser scanning microscopy, is employed to scan the thick biological tissue 10 and to capture image stack of the biological tissue incrementally.
In one embodiment, a laser 30 of the confocal microscopy 40 first scans the surface 1001 of the thick biological tissue 10 and then subsequently increases the focal depth of the confocal microscopy 40 incrementally to scan the tissue under the surface 1001, thereby capturing tissue images at different depths. The captured images are stacked to reconstruct a 3-dimensional image of the thick biological tissue 10. Because the tissue scatters the excitation and emission light travelling in the specimen, the deeper the scanning depth is, the worse the image definition will be.
As shown in
Next, a removal apparatus, such as mechanical slicer, laser cutter, chemical erosion, or cauterization, is used to remove part of the shallow portion 100 of the thick biological tissue 10 above the plane 1003 which is defined as removal plane, as shown in
As shown in
After executing several rounds of the foregoing tissue removal, images of multiple layers of the thick biological tissue 10 can be stacked to reconstruct a 3-dimensional image of the thick biological tissue 10. Although the thick biological tissue 10 experiences a plurality of cuttings (or removals), they do not interfere with the capture of tissue images because images of the shallow portion 100 are taken before the tissue removal is executed. Therefore, the reconstructed image is an intact 3-dimensional image of the thick biological tissue 10.
Although preferred embodiments of the present invention have been described, it will be understood by those skilled in the art that the present invention should not be limited to the described preferred embodiments. Rather, various changes and modifications can be made within the spirit and scope of the present invention, as defined by the following Claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 099133619 | Oct 2010 | TW | national |
