TWI761016B - Method for preparation of tissue sections - Google Patents

Abstract

Provided is a method for preparing a tissue section, including treating a tissue specimen with a clearing agent and at least one labeling agent to obtain a cleared and labeled tissue specimen; generating a three-dimensional (3D) image of the cleared and labeled tissue specimen; performing an image slicing procedure on the 3D image to generate a plurality of two-dimensional (2D) images; identifying a target 2D image among the plurality of 2D images to obtain a distance value of D1, which indicates the distance between the target 2D image and a predetermined surface of the 3D image; preparing a hardened tissue specimen from the cleared and labeled tissue specimen; and cutting the hardened tissue specimen near a predetermined site to obtain a tissue section, wherein the distance between the predetermined site and a surface of the hardened tissue specimen corresponding to the predetermined surface of the 3D image is D1.

Description

Preparation method of tissue slices

The present invention relates to a method for preparing a biological specimen, and more particularly, to a method for preparing tissue sections using a three-dimensional (3D) histopathology imaging method.

Histopathology refers to the microscopic examination of tissue to study the manifestations of disease. Histopathology can be more clearly defined and referred to in clinical medicine as the processing of a biopsy or surgical specimen and the placement of the tissue section on a glass slide before the specimen is examined by a pathologist .

Three-dimensional histopathological imaging involves the use of existing technologies, such as microscope systems and computerized imaging systems, to facilitate traditional microscopic examination. Figure 1 depicts a conventional technique for performing three-dimensional histopathology. As shown in FIG. 1, a tissue sample is obtained by biopsy. The tissue can be lung or kidney tissue. Thereafter, the tissue specimen is processed by the so-called formalin-fixed paraffin-embedded (FFPE) method to form a tissue block. Then, the tissue block was cut into slices, each slice having a thickness of about 3-5 μm. Next, each section was stained, and a camera-equipped microscope was used to image the sections and generate a two-dimensional image of each section. The 2D images can be sent to a computer and processed. The computer collects and processes the two-dimensional images to reconstruct a three-dimensional image therefrom. However, this conventional technique results in a 2 μm image loss between two consecutive 2D images because the tissue is sliced from the start.

Figure 2 depicts another conventional technique for obtaining three-dimensional histopathology images. Similarly, a tissue is obtained by biopsy, but then the tissue is stained and embedded in paraffin to form a tissue block. Subsequent slicing procedures, two-dimensional imaging and three-dimensional reconstruction procedures are similar to those described in FIG. 1 , so the relevant descriptions are omitted. Despite the improved performance of this technique, there is still an image loss of 1-2 μm between two consecutive 2D images, and this image loss is also attributed to the sectioning process. Therefore, how to reduce the image loss is still a problem to be solved.

Another problem stemming from traditional histopathology is that blind dissection during tissue section preparation may destroy the integrity of important morphological features in the tissue, thereby reducing the representativeness of the resulting tissue section to its source. Furthermore, a single biopsy specimen typically requires hundreds of cuts and hundreds of stainings to obtain hundreds of stained tissue sections for subsequent review, yet only a few of these sections show decisively abnormal results and can used as a basis for diagnosis. Obviously, in this case, excessive time and chemicals are consumed in preparing hundreds of tissue sections that cannot be used further. Therefore, it is necessary to develop a new tissue section preparation method to improve the accuracy and efficiency of clinical diagnosis.

An object of the present invention is to provide a method for preparing a tissue section directly from a specific part of a tissue specimen, which method utilizes a three-dimensional histopathological imaging method that reduces image loss. More specifically, the method comprises the steps of: (a) treating a tissue sample with a clarifying agent and at least one labeling agent for labeling a cellular component to obtain a clarified and labeled tissue sample; (b) generating the A three-dimensional image of the clarified and labeled tissue specimen; (c) performing an image slicing procedure on the three-dimensional image to generate a plurality of two-dimensional (2D) images; (d) from the plurality of two-dimensional (2D) images A target 2D image is identified in the 3D image to obtain a distance value D1, where D1 is the distance between the target 2D image and a predetermined surface of the 3D image; (e) from the clarified and marked tissue specimen preparing a hardened tissue specimen; and (f) cutting near a predetermined position of the hardened tissue specimen to obtain a tissue section, wherein the predetermined position is between a surface of the hardened tissue specimen corresponding to the predetermined surface of the three-dimensional image The distance is D1. Through the steps (a)-(c) constituting the three-dimensional histopathological imaging method, and the steps (d)-(f) of applying spatial information to solid cutting, the method of the present invention can efficiently produce more representative tissue slices.

In some embodiments, the clarifying agent is an aqueous clarifying agent, and the refractive index of the aqueous clarifying agent is 1.33-1.55, preferably 1.40-1.52, more preferably 1.45-1.52. Treatment with this clarifying agent can render a tissue specimen of at least 200 μm in thickness sufficiently transparent, while preventing tissue shrinkage or deformation, and avoiding lipid removal. Because the structural integrity of the clarified tissue specimen is well preserved, 3D imaging of the specimen provides more accurate morphological information. In addition, the fluorescent labeling of cell membranes and membrane-associated proteins is compatible with this clarifying agent, thus enabling the detection of various marker proteins of various diseases, especially of cancer.

In certain embodiments, the labeling agent is a fluorescent dye, or a conjugate of a fluorescent dye and a molecular probe selected from the group consisting of promoting A group consisting of agonist, antagonist, antibody, avidin, oligonucleotide, lipid nucleotide and toxin.

In certain embodiments, the tissue specimen in step (a) is embedded in an embedding material that provides physical support for the tissue specimen. The embedding material can be an agarose gel or a hydrogel.

In certain embodiments, the labeling agent is removed from the clarified and labeled tissue specimen prior to step (e), or removed from the tissue section after step (f). When the labeling agent is removed, the tissue section can be further stained to obtain a stained tissue section. Tissue staining can be performed by tissue staining methods well known in the art, such as hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), immunofluorescence (IF) )staining), and in situ fluorescence in situ hybridization (FISH) staining.

In certain embodiments, the clarifying agent and the marking agent are included in a clarifying composition, and the clarifying composition further includes a permeating agent. In the clarifying composition, the clarifying agent comprises a refractive index matching material selected from the group consisting of radiocontrast agents, monosaccharides, oligosaccharides, and any combination thereof 's group. The penetrant is preferably a surfactant, and the surfactant is selected from Triton X-100 (Triton X-100), polysorbate 20 (Tween-20), polysorbate 80 ( Tween-80), sodium dodecyl sulfate (SDS), n-dodecyl-β-D-maltoside (n-dodecyl-β-d-maltoside, DDM), urea (urea), 3-[3-(cholamidopropyl)-dimethylamine]-1-propanesulfonate (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, CHAPS), sodium deoxycholate (sodium deoxycholate), and any combination thereof.

In certain embodiments, the three-dimensional image is generated by scanning the clarified and labeled tissue specimen using a microscopy technique, such as laser scanning microscopy. The three-dimensional image is a three-dimensional model of the tissue specimen, and the three-dimensional image can be further analyzed in the three-dimensional dimension through the image slicing procedure. In the image slicing procedure, the 3D image is sliced in different directions to generate a plurality of 2D image slices. The aforementioned image slicing is performed along an axis selected from three mutually perpendicular axes (ie, an X-axis, a Y-axis, and a Z-axis) of the 3D image.

In certain embodiments, the labeled cellular component is a cell membrane, an organelle, or a biomolecule. The organelle can be a membranous organelle (eg, the nucleus), or a membraneless organelle (eg, the ribosome). The biomolecule can be a nucleic acid or a protein, such as a disease-related protein. The labeling step allows the structural or physiological state of the tissue specimen to be revealed at the cellular level and even the molecular level, thereby enabling identification of a target 2D image exhibiting a specific feature from a plurality of 2D images. In some embodiments, the target 2D image is identified by measuring the representation of a biomolecule in each 2D image of the plurality of 2D images. In certain embodiments, the target 2D image is obtained by measuring the proportion of cells with an abnormal pattern (eg, cell aggregation or cell invasion) relative to all cells in each of the plurality of 2D images and identified.

The methods disclosed herein can operate on a platform that includes a three-dimensional histopathology imaging system coupled to a microtome. The three-dimensional histopathology imaging system includes a microscope and a processor. The microscope system is set up to create a three-dimensional image of a tissue specimen. The processor is configured to perform an image slicing process on the 3D image to generate a plurality of 2D images, identify a target 2D image, and generate a distance value D1. The microtome may be a conventional microtome capable of cutting a predetermined location of the specimen.

The methods disclosed herein can directly prepare tissue sections that best represent a diseased or disease-prone tissue, thereby saving time and chemicals required to prepare tissue slides, as well as space required for slide storage. In addition, since the most representative parts of the tissue specimen can be identified before physical sectioning, these parts can be avoided from being damaged by blind cutting. Therefore, pathologists can make more accurate diagnoses based on tissue samples prepared by this method, and assist physicians in deciding the appropriate treatment for each patient.

Those skilled in the art will have a clear understanding of the present invention from the following detailed description of the preferred embodiment with reference to the accompanying drawings, in which: FIG. 1 depicts a conventional three-dimensional histopathology imaging method; FIG. 2 depicts another conventional 3D histopathological imaging method; Figure 3 is a flow chart describing the steps of the method disclosed herein; Figure 4a shows a virtual representation of a 3D image of a normal breast tissue specimen in the XY plane, XZ plane or YZ plane slice; Figure 4b shows a virtual slice of a 3D image of a breast cancer tissue specimen in the XY plane, XZ plane or YZ plane; Figure 5a is a 3D image of a lung tissue specimen; the specimen is marked with two fluorescent dyes to the nucleus and cell membrane respectively , and was also labeled with a primary anti-thyroid transcription factor 1 (TTF-1) antibody, which was recognized by a secondary antibody bound to a fluorescent dye; Figure 5b shows three batches of two-dimensional The amount of TTF-1 expression in each 2D image in the images, wherein each batch of 2D images is the XY plane, YZ plane or XZ plane (that is, along the Z axis, X axis, or Y-axis) at different depths sliced; Figure 5c shows a 2D image sliced along the XY plane and showing the highest TTF-1 expression; Figure 5d shows a slice along the YZ plane and showing the highest expression of TTF-1 Figure 6a is a 2D image obtained from a 3D image of a breast tissue specimen sliced at a depth of 30 μm below the top surface; Figure 6b is a 3D image from a breast tissue specimen with a depth below the top surface of 2D image sliced at 60 μm; Figure 6c is a 2D image sliced at a depth of 90 μm below the top surface of a 3D image of a breast tissue specimen; Figure 6d is a top 3D image from a breast tissue specimen 2D image sliced at a depth of 120 μm below the surface; Figure 6e is a 2D image sliced at a depth of 150 μm below the top surface of a 3D image of a breast tissue specimen; Figure 6f is a reproduction of Figure 6e, The stroma and tumor margin are outlined by dotted and dashed lines, respectively; and Figure 6g is a microscopic image of an H&E stained section containing the pathological features revealed in Figure 6e.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It can be further understood that these techniques Terms, such as terms defined in commonly used dictionaries, should be construed to have meanings consistent with their meanings in the relevant technical field and the context of this document, and unless explicitly defined herein, these terms are not intended to be idealized in an overly formal or overly formal sense.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in relation to the embodiment is included in at least one embodiment. Thus, appearances of the terms "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly defines otherwise.

Numerical values provided herein are approximations, and experimental values may vary within 20%, preferably within 10%, more preferably within 5%.

As shown in Figure 3, the methods disclosed herein comprise the steps of: (a) treating a tissue sample with a clarifying agent and at least one labeling agent for labeling a cellular component to obtain a clarified and labeled tissue sample; (b) ) generating a three-dimensional image of the clarified and labeled tissue specimen; (c) performing an image slicing procedure on the three-dimensional image to generate a plurality of two-dimensional images; (d) identifying a target two from the plurality of two-dimensional images 3D image to obtain a distance value D1, where D1 is the distance between the target 2D image and a predetermined surface of the 3D image; (e) preparing a hardened tissue sample from the clarified and labeled tissue sample; and ( f) Cutting near a predetermined position of the hardened tissue specimen to obtain a tissue section, wherein the distance between the predetermined position and a surface of the hardened tissue specimen corresponding to the predetermined surface of the three-dimensional image is D1.

The methods of the present invention begin with the collection of a tissue sample or specimen from an individual undergoing diagnosis. This system refers to a mammal, including humans and non-humans, such as primates, rodents, dogs, cats, cows, horses, rabbits, pigs, and the like. Whether the individual is already diseased or susceptible to disease, a biopsy, the process of removing a piece of tissue from an individual's body, is usually performed so that the specimen can later be analyzed to determine the presence and absence of disease degree. Various types of biopsies can be performed in order to obtain a tissue specimen for inspection in accordance with the methods disclosed herein. Examples of biopsies include skin biopsies, endoscopic biopsies, needle puncture biopsies, bone marrow biopsies, and surgical biopsies.

In step (a) of the methods disclosed herein, a tissue specimen is treated with a clarifying agent and at least one labeling agent for labeling a cellular component. This tissue clearing process makes a tissue optically clear or transparent by homogenizing its various refractive indices, thereby reducing light scattering and increasing light penetration. Therefore, tissue clarification allows thick tissue specimens with a thickness of about 150-250 μm to undergo almost no physical tissue sectioning before being observed under a microscope, thus reducing human biases that may occur due to tissue stretching, bending and tearing.

In some embodiments, the clarifying agent is an aqueous clarifying agent, and the refractive index of the aqueous clarifying agent is 1.33-1.55, preferably 1.40-1.52, more preferably 1.45-1.52. The clarifying agent includes a solvent and a refractive index (RI) matching material. The solvent may be water, phosphate buffered saline (PBS; eg 137 mM sodium chloride, 2.7 mM potassium chloride, 7.7 mM disodium phosphate and 1.47 mM potassium dihydrogen phosphate in water, pH 7.4) or other inorganic buffers . The aqueous clarifier can be prepared by adding a refractive index matching material to water or PBS, the refractive index matching material can be selected from glycerol, iodobenzene (sold under the name Histodenz), formamide (formamide), triethanolamine (triethanolamine), meglumine diatrizoate (meglumine diatrizoate) and any combination thereof. The final concentration of the refractive index matching material in the aqueous clarifying agent can vary between 30-70 wt%. Tissue clarification using such aqueous clarifiers can be performed at room temperature for 2 to 12 hours, preferably 2 to 8 hours, more preferably 2 to 4 hours.

In addition to being clarified, tissue samples are also labeled by at least one labeling agent for labeling a cellular component such as a cell membrane, nucleus, or having a specific amino acid sequence or a specific modification or a specific conformation of a protein. In certain embodiments, the labeling agent is a fluorescent dye, or a conjugate of a fluorescent dye and a molecular probe selected from the group consisting of promoters, antagonists, antibodies, A group consisting of avidin, oligonucleotides, lipid nucleotides and toxins. Different kinds of labeling agents are suitable for labeling different targets. For example, the nucleus can be treated with propidium iodide (PI), 4',6-diamidino-2-phenylindole (4',6-diamidino-2-phenylindole, DAPI) and SYTO series Stain (eg, SYTO 16 and SYTO 40 from Thermo Fisher Scientific), NucRed stain, or NucGreen stain. Membranous structures of cells can be labeled with lipophilic fluorescent stains, such as the Di series of stains (eg, DiD and DiR from Invitrogen), the PKH series of stains (eg, PKH26 and PKH67 from Merck). A marker protein related to a disease can be labeled with a primary antibody and a secondary antibody. The primary antibody can specifically bind to the marker protein, and the secondary antibody can recognize the primary antibody.

Step (a) can be carried out by contacting the tissue specimen with the clarifying agent and the labeling agent separately or simultaneously. In certain preferred embodiments, the clarifying agent and the labeling agent are included in a clarifying composition, and the clarifying composition further includes a penetrant such that tissue clarifying and tissue labeling can be accomplished in a single step. In the clearing composition, the refractive index matching material is preferably selected from the group consisting of radiographic contrast agents (eg, iodixanol), monosaccharides (eg, fructose), oligosaccharides (eg, sucrose), and any combination thereof group. The penetrant is preferably a surfactant, and the surfactant is selected from tritraton X-100, polysorbate 20, polysorbate 80, sodium dodecyl sulfate (SDS), n-dodecyl sulfate Alkyl-β-D-maltoside (DDM), urea, 3-[3-(cholamidopropyl)-dimethylamine]-1-propanesulfonate (CHAPS), sodium deoxycholate and its A group of any combination. The concentration of the refractive index matching material may be between 30% and 80% (w/v). The concentration of the penetrant may be between 0.1% and 2% (v/v). The concentration of the labeling agent can range from 100 ng/ml to 1 mg/mL.

Tissue specimens for clarification and labeling steps can be fresh or stocked. In one embodiment, the tissue specimen is immediately collected from a portion of a patient's body (eg, a solid tumor) and is therefore in a fresh state. In another embodiment, the tissue specimen is processed according to sample preparation methods known to those skilled in the art, and is referred to as a stock specimen. The sample preparation method may include the steps of fixing, dehydration, infiltration and embedding. Fixation is a procedure to prevent decay and maintain tissue morphology. In this procedure, a tissue specimen is immersed at room temperature in a fixative, such as formalin (4% by mass formaldehyde in buffered saline), for a duration of typically 4-48 hours, depending on the size of the tissue specimen different. Dehydration is a procedure to remove water from a fixed specimen by treating it with increasing concentrations of dehydrating agents. For example, 70%, 95% and 100% alcohols such as ethanol are applied, and then treated with xylene. Infiltration is the procedure of infiltrating an embedding medium, such as resin or wax, into the specimen. An example of infiltration is placing a dehydrated specimen in a mixture of xylene and a molten wax (eg, paraffin wax heated to 56-60°C). Embedding is performed by transferring an infiltrated specimen into an embedding vessel, whereupon an embedding medium (eg, molten paraffin) is introduced around the specimen and allowed to cool to form a tissue mass (also known as a tissue mass). ). Formalin-fixed and paraffin-embedded stock specimens are referred to as FFPE-treated.

Where the tissue specimen is fresh, the specimen can be further fixed with a fixative such as formalin for 6 to 12 hours at room temperature prior to the clarification and labeling steps. Alternatively, when the specimen to be examined is FFPE-treated, it can be dewaxed with xylene and alcohol at room temperature for 2 hours and 4-6 hours, respectively, before the clarification and labeling steps.

During the tissue clarification and labeling process of step (a), the tissue specimen can be embedded in an embedding material, such as an agar gel or a hydrogel. Embedding can provide physical support for the specimen. In one embodiment, the agar gel is prepared from a warm aqueous solution containing 1-4% w/w agarose. In another embodiment, the hydrogel is prepared from an aqueous dispersion of at least one natural or synthetic polymer that cures upon changes in temperature, pH, salinity, or radiation. Examples of such polymers include alginates, hyaluronates, and acrylamide-based polymers.

In the next step (b), a three-dimensional image of the clarified and labeled tissue specimen is generated by microscopy. In some embodiments, a scanning laser microscope is used to obtain a plurality of consecutive 2D images of the clarified and labeled tissue specimen at different depths, and a 3D image can be reconstructed from the images. Examples of scanning laser microscopes include laser scanning confocal microscopy (LSCM), two-photon microscopy (two-photon microscopy), three-photon microscopy (three-photon microscopy), and line scanning conjugate microscopy Line-scanning confocal microscopy. Other microscopes involving scanning techniques such as spinning disk confocal microscopy or light-sheet microscopy can also be applied to the methods disclosed herein to generate three-dimensional images of tissue specimens. Therefore, the scope of the present invention should not be limited to scanning laser microscopy techniques. Since the scanning procedure is performed on an intact tissue specimen, in other words, the tissue specimen is scanned before being cut into slices, the three-dimensional images obtained by the methods disclosed herein have less image loss.

In the next step (c), an image slicing procedure is performed on the 3D image to generate a plurality of 2D images. The image slicing process is a virtual slicing process, during which the 3D image is sliced into a plurality of virtual slices. Each image slice can be presented on a display like a conventional two-dimensional image, showing a cross-sectional view of the imaged tissue specimen. Virtual image slices are well known in the field of image processing, so for the sake of convenience, related descriptions about virtual image slices will be omitted.

The virtual slice of the 3D image can be along the X-axis, Y-axis, Z-axis or a combination thereof. In some embodiments, the 3D image is sliced sequentially along three axes to generate three batches of 2D images (ie, multiple 2D images in the XY plane, the YZ plane, and the XZ plane). The aforementioned X-axis, Y-axis and Z-axis can be interpreted as coordinate axes in a Euclidean space, and the three-dimensional image can be represented as a vector in the Euclidean space. In some embodiments, the 3D image is virtually sliced along the three axes to generate a plurality of 2D images on the XY plane, the YZ plane, and the XZ plane. The X, Y and Z axes The direction of , is not particularly limited, as long as the X-axis, Y-axis, and Z-axis are perpendicular to each other. In other words, the X-axis, Y-axis and Z-axis together constitute an orthogonal set (each a unit vector) that can be rotated arbitrarily.

In some embodiments, the three-dimensional image is sliced into different slices to generate a plurality of two-dimensional images with biometric profiles. The biological profile can be a profile of the presentation of a biomolecule, a staging profile of histomorphological abnormalities, or a profile of other biological features. In certain embodiments, the biological characteristic is a disease characteristic that can be quantified and assessed. For example, the biological signature can be the expression level of a characteristic protein of a cancer, the TNM stage established by the American Joint Committee on Cancer (AJCC), or determinable using analytical methods known in the field of clinical medicine. Tumor invasion status.

Diseased tissue is heterogeneous, especially tumor/cancer tissue. Heterogeneity makes the diseased tissue look distinctly different from different directions. Please refer to Figures 4a and 4b, which depict virtual slices of a 3D image in different directions. Figure 4a shows, from left to right, sectioning of a normal breast tissue specimen in the direction of the XY plane, the XZ plane, or the YZ plane. The normal breast tissue specimen was collected from a healthy individual and was further clarified and labeled with fluorescent dyes used to label cell membranes and nuclei. Figure 4b shows, from left to right, sectioning of a breast cancer tissue specimen in the direction of the XY plane, the XZ plane, or the YZ plane. The breast cancer tissue specimen was collected from a breast cancer patient, and was further clarified and labeled with fluorescent dyes used to label cell membranes and nuclei. It can be clearly seen from Figure 4b that the three-dimensional images of breast cancer tissue observed from the XY plane, XZ plane, or YZ plane are quite different. In contrast, in Figure 4a, a three-dimensional image of normal breast tissue viewed from the XY plane, the XZ plane, or the YZ plane shows a similar pattern.

In the next step (d), a target 2D image is identified from the plurality of 2D images with respective biological characteristic profiles. Since in a batch of 2D images, a specific 2D image can be defined by a distance value D1, which refers to the distance between the target 2D image and a predetermined surface of the 3D image, the The identification of the target 2D image can yield a definite D1 value. The D1 value not only indicates the position of the target 2D image in the 3D image, but also indicates the position of a specific part corresponding to the target 2D image in the tissue sample. Therefore, the D1 value can be used to assist in the preparation of a specific tissue section from a tissue specimen.

Please refer to Figure 5a, which is a three-dimensional image of a human lung tissue specimen. The specimen is clarified and then labeled with a fluorescent dye for nuclei (shown in red), a fluorescent dye for cell membranes (shown in blue), and an anti-thyroid transcription factor 1 (shown in blue). TTF-1) antibody, which was Recognized by a secondary antibody (shown in green) that binds Alexa Flour 555, a fluorescent dye. TTF-1 is commonly used as a predictive and prognostic marker in the diagnostic evaluation of suspected lung cancer cases. Lung cancer can be divided into the following four grades: grade 1 (also denoted +), with 6% to 25% of tumor cells positive for TTF-1; grade 2 (also denoted ++), with 26 % to 50% of tumor cells were TTF-1 positive; grade 3 (also denoted +++), 51% to 75% of tumor cells were TTF-1 positive; and grade 4 (also denoted ++) ++), more than 75% of tumor cells were positive for TTF-1.

To identify and further evaluate biological features in a 3D image, such as protein expression or tumor region, a straightforward approach is to use 3D feature recognition algorithms, such as 3D convolutional neural network , CNN). However, executing such algorithms involves expensive computing infrastructure, which limits the application of 3D image analysis. In addition, such algorithms can only identify certain features in a 3D image, but cannot capture the most representative 2D image that is sufficient for clinical diagnosis. Therefore, in the field of clinical diagnosis, it is more appropriate to analyze 2D image slices rather than 3D images using feature recognition algorithms based on 2D images.

The 3D image in Figure 5a is virtually sliced into multiple batches of multiple 2D images in the direction of the XY, XZ, or YZ plane, and the TTF- 1 The amount of expression was determined by the ratio of the number of TTF-1 positive cells to the total number of cells. A specific 2D image in a specific batch of 2D images can be defined by a distance value D1 between the 2D image and a predetermined surface of the 3D image. For example, D1 may be the vertical depth (μm) of a 2D image below the top surface of a 3D image. As shown in Figure 5b, the complex 2D images sliced at different depths in the YZ plane have different TTF-1 representations (expressed as percentages), and the 2D images with the highest TTF-1 representation can be Identified as being located 197 μm below the top surface of the 3D image. Likewise, among the complex 2D images sliced at different depths in the XY or XZ plane directions, the 2D images with the highest TTF-1 performance can be identified. The related results are shown in Table 1.

According to Table 1, the average expression level of TTF-1 in different batches of 2D images was similar, but the highest expression level of TTF-1 was significantly different, showing the heterogeneity of tumor tissue. In addition, the three 2D images with the highest expression of TTF-1 were confirmed to show the morphology of lung cancer through microscopic examination of the tissue specimen by the pathologist. Therefore, any one of the three 2D images can be selected as the target 2D image. Preferably, a 2D image sliced at a depth of 197 μm (ie D1 value) in the YZ-plane (ie along the X axis) direction is selected as the target 2D image.

When a target 2D image is identified and the D1 value is obtained, a tissue section containing the structural features revealed by the target 2D image can be cut from the clarified and labeled tissue specimen. According to steps (e) and (f) of the methods disclosed herein, the clarified and labeled tissue specimen is processed to form a hardened tissue specimen that does not deform when sectioned. Subsequently, the incision is performed in the vicinity of a predetermined position of the hardened tissue specimen, the predetermined position being varied depending on the value of D1. More specifically, the predetermined position is separated by a distance D1 from a surface of the hardened tissue specimen corresponding to the predetermined surface of the three-dimensional image. An example of such a corresponding surface is the top or bottom surface of a hardened tissue sample.

The hardened tissue specimen can be prepared, but is not limited to, by embedding the tissue specimen in an embedding medium, such as paraffin, or by cooling the tissue specimen at a temperature below -5°C. The hardened tissue specimen is then moved to a microtome for sectioning near a predetermined location. The slicing machine is a slicing device for making micron-scale material flakes. A conventional microtome includes an adjustable sample holder that holds the specimen and adjusts the position and orientation of the specimen; a movable blade that can be moved to an optimal sectioning position relative to the specimen; and operational components that support other functions, Other functions such as setting slice thickness and mechanical trimming. In certain embodiments, a conventional microtome is used to slice predetermined locations of the hardened tissue specimen to produce a tissue section. In other embodiments, the hardened tissue specimen is serially sectioned from 1-15 μm above the predetermined position to 1-15 μm below the predetermined position to generate at least two or more tissue sections, which are After staining and examination by a pathologist, one of the most representative tissue sections can be selected.

The methods disclosed herein may further comprise removing a labeling agent from the clarified and labeled tissue specimen prior to step (e), or removing a labeling agent from the tissue section after step (f). When the labeling agent is removed after step (f), it can be done by soaking the tissue section in stripping buffer way to remove the marker. An example of such a removal buffer is Tris buffer containing 0.8% 2-mercaptoethanol and 2% SDS. This soaking procedure can be performed at room temperature or higher. In a preferred embodiment, the tissue sections are first deparaffinized with xylene (or D-limonene sold under the name Hemo-De) and alcohol, and then rinsed with PBS at room temperature. Tissue sections. Thereafter, the tissue section was immersed in a removal buffer at about 56°C for 45 minutes, and washed with PBS.

The methods disclosed herein can further comprise staining the tissue section to obtain a stained tissue section after the labeling agent is removed. Tissue staining can be performed by staining methods well known in the art. Examples of staining methods include, but are not limited to, H&E staining, immunohistochemistry (IHC), immunofluorescence (IF) staining, and fluorescence hybrid in situ (FISH) staining.

In one embodiment where immunohistochemistry is performed, staining of tissue sections is performed first with a primary antibody for about 12 hours at 4°C, followed by a secondary antibody conjugated to a chromogenic enzyme at room temperature Do this for about 30 minutes. Thereafter, the tissue sections were soaked in a chromogen solution for about 5 minutes at room temperature. Finally, the tissue sections were counterstained with hematoxylin for about 4 minutes at room temperature and washed with distilled water.

In summary, the method disclosed herein utilizes a 3D histopathology imaging method with reduced image loss to generate a high-fidelity 3D image of a tissue specimen, and utilizes virtual image slicing and 2D image-based imaging before physical slicing. A feature identification algorithm is used to analyze the structure of the tissue specimen, and finally a tissue section containing pre-identified histopathological features is obtained from the tissue specimen. Given that the 3D image generation process in the disclosed method does not involve cutting the tissue specimen into segments, little or no image loss can be expected. In addition, analysis of 2D images sliced along multiple axes can yield rich information that enables healthcare workers to make more reliable diagnostic decisions and make more appropriate treatment recommendations. For example, physicians can rely on a profile of human epidermal growth factor receptor 2 (HER2) expression obtained from 2D imaging to determine whether a patient is eligible for treatment with Herceptin (trastuzumab). conditions of. Accurate assessment of HER2 status can ensure that only patients who would benefit from treatment with Heaiping will receive it.

The methods disclosed herein can operate on a platform that includes a three-dimensional histopathology imaging system coupled to a microtome. The three-dimensional histopathology imaging system includes a microscope and a processor. The microscope may be a conventional microscope capable of creating a three-dimensional image of a tissue specimen. The processor can be any device capable of executing computer instructions. In particular, the processor is configured to perform an image slicing process on the 3D image to generate a plurality of 2D images, identify a target 2D image, and And output a distance value D1. The processor may be, but is not limited to, a microprocessor, a microcontroller or a central processing unit (CPU). The processor may be included in a computer that further includes a memory unit for storing computer instructions. The microtome may be a conventional microtome capable of cutting a predetermined location of the specimen.

Example 1

Preparation of breast tissue sections from breast tissue specimens

Collect a fresh breast tissue sample from a female patient with possible breast cancer. The tissue specimens were first fixed with 4% formaldehyde and then infiltrated with 0.1-1% Triton-100. Subsequently, the tissue specimens were stained with SYTO 16 and DiD to label cell nuclei and cell membranes, respectively. Each of the labeling was performed at room temperature for 8 hours. Next, the labeled specimen was immersed in an aqueous clarifying agent at room temperature for about 8 hours. The clarified and labeled tissue specimen, which had a thickness of about 150 μm, was imaged using a scanning laser conjugation microscope system (LSM780; Zeiss) from the top surface to the bottom surface of the specimen to obtain about 100 Å of the specimen. consecutive 2D images are then used to generate a 3D image of the specimen. The lateral resolution (in the X and Y directions) of the images was less than 1 μm, and the axial resolution (in the Z direction) was less than 2 μm.

The 3D image is virtually sliced along the Z axis to generate a batch of 2D images, which are then reviewed by a pathologist to identify images showing stroma adjacent to the tumor margin. This feature is of interest because clinical studies have shown that the morphology of the stroma correlates with the risk of cancer cell metastasis. Figures 6a-6e show 2D images sliced at depths of 30 μm, 60 μm, 90 μm, 120 μm and 150 μm below a top surface of the 3D image, respectively. Figure 6f is a reproduction of Figure 6e, in which the stroma and tumor margins are outlined by dotted and dashed lines, respectively. In these images, the stroma was only visible in Figure 6e. Therefore, Figure 6e is identified as the target 2D image. Since FIG. 6e is located at a depth of 150 μm below the top surface of the 3D image, the distance value D1 is set to be 150 μm from the top surface of the 3D image.

Subsequently, the specimen was dehydrated and embedded in paraffin, followed by serial sectioning of the embedded specimen using a slide microtome (pfm Slide 2003) at the top surface of the embedded specimen (corresponding to the The top surface of the 3D image) is approximately 150 μm below the depth. Perform the slicing action 3-7 times to obtain a plurality of tissue sections with a thickness of 5 μm, take one of the tissue sections, prepare a stained tissue section according to the known H&E staining method in the art, and use the MoticEasyScan digital slide to scan camera (Motic, San Francisco, USA). Figure 6g is a micrograph of the H&E stained section picture. Notably, Figures 6g and 6e are histomorphologically similar, illustrating that the methods disclosed herein can be used to directly prepare tissue sections with previously identified histopathological features.

Claims (12)

A method for preparing a tissue section, comprising the steps of: (a) treating a tissue specimen with a clarifying agent and at least one labeling agent for labeling a cell component to obtain a clarified and marked tissue specimen, wherein the clarifying agent is a is an aqueous clarifying agent having a refractive index of 1.33-1.55; (b) generating a three-dimensional image of the clarified and labeled tissue specimen, wherein the three-dimensional image is obtained by scanning the clarified and labeled tissue specimen using a microscopy technique generating; (c) performing an image slicing procedure on the 3D image to generate a plurality of 2D images; (d) identifying a target 2D image from the plurality of 2D images to obtain a distance value D1, wherein D1 is for the distance between the target two-dimensional image and a predetermined surface of the three-dimensional image; (e) preparing a hardened tissue specimen from the clarified and labeled tissue specimen; and (f) near a predetermined location of the hardened tissue specimen Cutting is performed to obtain a tissue section, wherein the distance between the predetermined position and a surface of the hardened tissue specimen corresponding to the predetermined surface of the three-dimensional image is D1. The method of claim 1, wherein the marker is a fluorescent dye, or a conjugate of a fluorescent dye and a molecular probe selected from the group consisting of promoters, antagonists , antibodies, avidin, oligonucleotides, lipid nucleotides and toxins. The method of claim 1, wherein in step (a) the tissue specimen is embedded in an embedding material. The method of claim 1, further comprising removing the marker from the tissue section after step (f). The method of claim 4, further comprising staining the tissue section to obtain a stained tissue section after the labeling agent is removed. The method of claim 1, wherein in the image slicing procedure, slicing the 3D image is performed along an axis selected from three mutually perpendicular axes of the 3D image. The method of claim 1, wherein the cell component is a cell membrane, an organelle or a biomolecule. The method of claim 7, wherein the target 2D image is identified by measuring the amount of representation of the biomolecule in each 2D image of the plurality of 2D images. The method of claim 1, wherein the target 2D image is identified by measuring the proportion of cells with abnormal patterns relative to all cells in each of the plurality of 2D images. The method of claim 1, wherein the clarifying agent and the marking agent are included in a clarifying composition, and the clarifying composition further includes a penetrant. The method of claim 10, wherein the clarifying agent comprises a refractive index matching material selected from the group consisting of radiocontrast agents, monosaccharides, oligosaccharides, and any combination thereof. The method of claim 10, wherein the penetrant is a surfactant, and the surfactant is selected from Triton X-100, polysorbate 20, polysorbate 80, dodecyl Sodium sulfate (SDS), n-dodecyl-β-D-maltoside (DDM), urea, 3-[3-(cholaminopropyl)-dimethylamine]-1-propanesulfonate (CHAPS) ), sodium deoxycholate, and any combination thereof.

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