WO2017193700A1 - 快速获取大样本三维结构信息和分子表型信息的成像装置和方法 - Google Patents

快速获取大样本三维结构信息和分子表型信息的成像装置和方法 Download PDF

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WO2017193700A1
WO2017193700A1 PCT/CN2017/077160 CN2017077160W WO2017193700A1 WO 2017193700 A1 WO2017193700 A1 WO 2017193700A1 CN 2017077160 W CN2017077160 W CN 2017077160W WO 2017193700 A1 WO2017193700 A1 WO 2017193700A1
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sample
imaging
slice
dimensional structure
module
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PCT/CN2017/077160
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English (en)
French (fr)
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龚辉
袁菁
骆清铭
江涛
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华中科技大学
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    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T15/00—3D [Three Dimensional] image rendering
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T15/00—3D [Three Dimensional] image rendering
    • G06T15/005—General purpose rendering architectures
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/10—Image acquisition modality
    • G06T2207/10072—Tomographic images
    • G06T2207/10101—Optical tomography; Optical coherence tomography [OCT]
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
    • G06T2207/00—Indexing scheme for image analysis or image enhancement
    • G06T2207/30—Subject of image; Context of image processing
    • G06T2207/30004—Biomedical image processing
    • G06T2207/30016—Brain

Definitions

  • the present invention relates to microscopic imaging, and in particular to an imaging apparatus and method for rapidly acquiring large sample three-dimensional structural information and molecular phenotypic information.
  • the brain is one of the most complex systems in nature, and it governs all human activities.
  • the exploration of the brain has always been the goal of human research, but to this day we still cannot accurately describe the mechanisms of intelligence, thinking and consciousness.
  • the neural structure is an important basis for achieving advanced brain function.
  • Complex brain function requires the participation of multiple brain regions and is coordinated by local and long-range neural circuits.
  • Brain diseases are often accompanied by structural abnormalities associated with specific neural circuits related to function and their input connections and output projections. In order to decipher the structural basis of brain function and brain disease, it is necessary to perform cell-resolved horizontal structural analysis at the whole brain scale.
  • understanding brain function and brain disease also needs to analyze its molecular basis, and define the types of neuronal cells involved in the loop and function in order to find out the molecular mechanism of disease occurrence and development.
  • neuron functional molecules there are many kinds of neuron functional molecules, and there are hundreds of categories identified at present, which directly leads to the complexity of neuronal molecular phenotypes.
  • neuron classification was performed only on a single molecular phenotype, which was not accurate enough. Therefore, finding the characteristic molecular phenotype to determine the type of neurons in the loop requires a large number of screening and identification at the whole brain scale, and the workload is huge.
  • the complicated manual operation also causes a certain amount of brain slices to be depleted, and it is impossible to obtain continuous neurological projection information at the whole brain level. Since the spatial position of adjacent slices cannot be registered, the acquired data set cannot reconstruct the three-dimensional structure. Therefore, the molecular phenotype of neural structures There is an urgent need to develop automated whole brain imaging methods.
  • the existing whole brain immunohistochemistry is only applicable to small molecule antibodies, and as the molecular weight of the antibody increases, the penetration depth and uniformity of the antibody are drastically decreased.
  • the whole brain immunohistochemistry process is long and complicated, the fluorescence signal is easy to quench, the sample is deformed, and it cannot be preserved for a long time.
  • the antibody reagent is used in a large amount and at a high cost.
  • the current optical illumination technology has a low imaging resolution of only about 10 ⁇ m, and further declines in the deep brain region, and it is impossible to obtain a consistent imaging effect in the whole brain range.
  • the ultra-long working distance objective lens required for imaging is difficult to design and expensive. There are currently no techniques for phenotypic staining of other whole brain molecules such as whole brain in situ hybridization.
  • the present invention aims to overcome the deficiencies of the prior art and provide an apparatus and method for quickly acquiring large sample three-dimensional structure information and molecular phenotype information.
  • the invention solves the complicated sample preparation process in the prior art, affecting sample morphology and fluorescence.
  • the shortcomings of signal and imaging speed can quickly acquire and analyze the three-dimensional structure information of the sample.
  • the acquired data has self-registration characteristics, and molecular phenotypic staining is performed on the sample slices of the part of interest to obtain molecular phenotypic information of the sample. It can be registered to the acquired three-dimensional structure information.
  • the technical solution adopted for achieving the object of the present invention is an imaging device for quickly acquiring large sample three-dimensional structure information and molecular phenotype information, the device comprising:
  • a three-dimensional mobile station for driving the sample storage device to move in a three-dimensional space
  • a vibration slicing module for slicing a sample to obtain a shallow portion of the sample
  • Wide field optical microscopy imaging module for high-throughput tomography of the shallow portion of the sample.
  • the present invention also provides a method for quickly acquiring large sample three-dimensional structure information and molecular phenotype information by the above device, the method comprising:
  • Step S101 labeling the biological tissue sample with a fluorescent marker
  • Step S102 embedding the sample with agarose to obtain the sample block after embedding
  • Step S103 fixing the embedded sample block in a water tank and adding a buffer
  • Step S104 setting a Z-direction sampling pitch, a slice thickness, an imaging interval, and a Z-direction imaging range in the computer according to imaging requirements;
  • Step S105 controlling the movement of the three-dimensional translation stage by the computer, moving the sample to the imaging area, and controlling the wide-field optical microscopic imaging module to perform high-throughput tomography on the entire shallow section of the sample, and storing the obtained image;
  • Step S106 The computer controls the movement of the three-dimensional translation stage, moves the sample to the slice area, and controls the vibration slice module to quickly slice the imaged portion of the sample;
  • Step S107 The computer controlled slice collection module collects the slice into the container
  • Step S108 confirm whether the imaging of the entire Z direction of the sample has been completed, and if not completed, perform the next step;
  • Step S109 the computer controls the three-dimensional translation stage to raise the sample in the Z direction, and then returns to step S105; if completed, performs the next step;
  • Step S110 The acquired images have self-alignment, and the imaging data of the sample three-dimensional structure information can be quickly reconstructed and browsed, and the sample slices of the region of interest are selected;
  • Step S111 performing molecular phenotypic staining on the selected sample slice and imaging, obtaining molecular phenotype information of the corresponding part, and registering to the existing three-dimensional structural information imaging data.
  • the sample slice generated in the automated collection imaging process can select the target slice of interest for molecular phenotypic staining according to the imaging result of the sample three-dimensional structure information, and obtain the molecular phenotype information of the sample. Avoiding the limitations of whole brain immunohistochemistry using only small molecule antibodies, It can be applied to any conventional molecular phenotypic staining reagents such as any immunohistochemical antibody and in situ hybrid antibody.
  • FIG. 1 is a schematic structural view of an apparatus for directly acquiring large-sample three-dimensional structure information and molecular phenotypic information according to the present invention.
  • FIG. 2 is a block diagram of a control connection of a computer in the present invention.
  • FIG. 3 is a flow chart of a method for rapidly acquiring large sample three-dimensional structure information and molecular phenotypic information imaging according to the present invention.
  • FIG. 4 is a schematic diagram of the present invention for quickly acquiring three-dimensional structure information of a large sample.
  • Fig. 5 is a three-dimensional structure data diagram of a mouse brain sample obtained in the present invention
  • Fig. 5a is a three-dimensional reconstruction result of whole brain structure data
  • Fig. 5b is a single coronal plane diagram.
  • FIG. 6 is a diagram showing immunohistochemical data of a sample section obtained in the present invention
  • FIG. 6a is an immunohistochemical map of anti-small albumin
  • FIG. 6b is an immunohistochemical map of anti-calcium binding protein.
  • the structure of the imaging device for quickly acquiring large sample three-dimensional structure information and molecular phenotypic information in this embodiment is shown in FIG. 1.
  • the device comprises a precision three-dimensional translation stage 9, a water tank 7, a wide field optical microscopic imaging module, a vibration slice module, and a slice.
  • the module and computer 15 are collected.
  • the water tank 7 is provided on a precision three-dimensional translation stage 9, in which a buffer solution is placed, and the sample 5 is placed in a buffer.
  • the precision three-dimensional translation stage 9 is connected to a computer 15 which controls the precise three-dimensional translation stage 9 to move in three dimensions.
  • the wide field optical microscopy imaging module used in this embodiment is a structured light illumination microscope for performing high-throughput tomography on a shallow portion of a sample, which includes a light source 1, a spatial light modulator 2, an objective lens 3, and a camera 4.
  • the spatial light modulator 2 is for modulating the light emitted by the light source
  • the objective lens 3 is for forming a structured light modulation stripe on the light modulated by the spatial light modulator, that is, the light emitted by the light source 1 passes through the space.
  • structured light modulation stripes are formed on the focal plane of the objective lens 3.
  • the camera 4 is used to acquire images of different phases and transmit them to the computer 15.
  • the tomographic images of the samples can be obtained by calculating three images of different phases through a structured light illumination imaging reconstruction algorithm.
  • the vibrating section module 8 used in this embodiment is used to cut off the imaged portion of the sample, and the vibrating section module 8 is an existing common device, and details are not described herein again.
  • the blade 6 of the vibrating section module 8 is located below the buffer surface in the water tank 7.
  • the slice collection module used in this embodiment is used to collect the excised slice, which comprises a container 12, a tube 10 and a water pump 11, one end of which is opposite the blade 6 (the portion where the slice is cut) and the other end leading to the container. 12; A water pump 11 is provided in the line 10 for drawing the buffer in the water tank 7 into the container 12.
  • the computer 15 is connected to the spatial light modulator 2, the camera 4, the vibration slicing module 8, the precision three-dimensional translation stage 9, and the water pump 11, respectively.
  • the computer 15 controls the above components separately: the computer 15 realizes structured light microscopic imaging by controlling the spatial light modulator 2 and the camera 4, acquires a tomographic image of the sample and stores it in the computer 15; the computer 15 controls the vibration slicing module 8 and the precision three-dimensional translation stage 9 realizes rapid slicing of the sample 5; the computer 15 draws the buffer in the water tank 7 into the container 12 by controlling the water pump 11, and the cut slice is also drawn into the container 12 with the buffer.
  • the computer 15 realizes structured light microscopic imaging by controlling the spatial light modulator 2 and the camera 4, acquires a tomographic image of the sample and stores it in the computer 15; the computer 15 controls the vibration slicing module 8 and the precision three-dimensional translation stage 9 realizes rapid slicing of the sample 5; the computer 15 draws the buffer in the water tank 7 into the container 12 by controlling
  • the light source 1 is an X-cite exact metal halide light source manufactured by Lumen Dynamics
  • the spatial light modulator 2 is a digital micromirror array having a size of 0.7 XGA
  • the objective lens 3 for imaging is used.
  • the method for quickly obtaining the large sample three-dimensional structure information and the molecular phenotype information by using the embodiment is as shown in FIG. 3, and specifically includes the following steps:
  • Step S101 labeling a specific structure of the biological tissue sample by using a fluorescent labeling technique
  • Step S102 Embedding the sample with agarose to obtain an embedded sample block.
  • the fresh sample tissue is fixed, it is embedded in 3% to 5% agarose, and the embedding process takes only 1 to 2 hours.
  • Step S103 Fix the embedded sample in the water tank 7 and add a sodium borate buffer.
  • Step S104 setting a Z-direction sampling pitch, a slice thickness, an imaging interval, and a Z-direction imaging range on the computer 15 according to imaging requirements;
  • Step S105 The computer controls the precise three-dimensional translation stage 9 to move the sample to the imaging area, and controls the structured light illumination microscope to perform high-throughput tomography on the shallow portion 13 of the sample 5.
  • the camera 4 obtains images of different phases and is passed by the computer.
  • the reconstruction algorithm calculates a tomographic image of the sample; and stores the obtained image;
  • Step S106 The computer controls the precise three-dimensional translation stage 9 to move the sample to the slice area, and controls the vibration slice module to perform rapid slice on the shallow imaged portion 13 of the sample;
  • Step S107 The computer controlled slice collection module collects the slice into the designated container 12;
  • Step S108 confirm whether the imaging of the entire Z direction of the sample has been completed, and if not completed, perform the next step;
  • Step S109 lifting the sample in the Z direction, returning to step S105, continuing to image and slice the exposed shallow portion 14 of the sample; if completed, performing the next step;
  • Step S110 The acquired images have self-alignment, and the imaging data of the sample three-dimensional structure information can be quickly reconstructed and browsed, and the sample slices of the region of interest are selected;
  • Step S111 performing molecular phenotypic staining on the selected sample slice and imaging, obtaining molecular phenotype information of the corresponding part, and registering to the existing three-dimensional structural information imaging data.
  • the process of quickly acquiring the three-dimensional structure information of the large sample by the above method is as follows: the wide-field optical microscopic imaging module performs high-throughput tomography on the shallow portion 13 of the sample 5 through the objective lens 3, after the imaging is completed.
  • the vibrating section module 8 cuts off the sampled imaged portion 13, and the slice collecting module collects the slice, and then the computer 15 controls the precision three-dimensional translation stage 9 to raise the sample in the Z direction, and continues to image the shallow portion 14 of the sample, and the shallow portion is formed after the imaging is completed.
  • the 14 sample, tomographic images and slices of the entire sample can be acquired by 14 excision, sectioning, collection, and imaging cycles.
  • FIG. 5 is a three-dimensional structural data diagram of a mouse brain sample obtained by the above-described imaging method for quickly acquiring large sample three-dimensional structure information and molecular phenotypic information.
  • the mouse brain was labeled by transgenic fluorescent labeling technology, and the cells containing the corticotropin releasing hormone gene expressed fluorescent protein in the brain, wherein FIG. 5a is a three-dimensional reconstruction result of the whole brain structure data, and FIG. 5b is a single graph. Coronal picture.
  • the entire set of data consists of 300 layers of images with 50 ⁇ m spacing between each layer of image. The resolution of the image reaches 0.32 ⁇ m ⁇ 0.32 ⁇ m, which can clearly distinguish details such as cell body and fiber.
  • the images have self-registration characteristics, which can easily obtain 3D reconstruction results and identify the distribution and aggregation of cells containing the corticotropin releasing hormone gene in the whole brain.
  • the acquisition time of the entire whole brain structure data takes only 12 hours.
  • FIG. 6 is an immunohistochemical data map of a cortical part slice of a rat brain sample obtained by the above-described imaging method for rapidly acquiring large sample three-dimensional structure information and molecular phenotypic information, wherein FIG. 6a is an immunohistochemical image of anti-small albumin.
  • Figure 6b is an immunohistochemical picture of anti-calcium binding protein. According to the results of the existing three-dimensional structure data, it is known that there are many cells containing the corticotropin releasing hormone gene in the cortex, so the sample containing the cortex is selected for immunohistochemical staining.
  • the staining operation is a routine operation step, which is simple and rapid, and can be applied to any conventional molecular phenotypic staining reagent such as any immunohistochemical antibody or in situ hybrid antibody.
  • the immunohistochemical data in the figure show the distribution of cells containing small albumin and calcium binding protein in the rat cerebral cortex.

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Abstract

一种快速获取大样本三维结构信息和分子表型信息的成像装置和方法，该装置包括：样本存放装置；用于驱动所述样本存放装置在三维空间内移动的三维移动台（9）；用于对样本（5）进行切片，使样本得到浅层部分的振动切片模块（8）；以及用于对样本的浅层部分进行高通量层析成像的宽场光学显微成像模块。该装置和方法解决了现有技术中样本制备流程复杂、影响样本形态和荧光信号、成像速度慢的缺点，能够快速获取并分析样本的三维结构信息，所获取数据具有自配准特性，并对感兴趣部位的样本切片进行分子表型染色，获得样本的分子表型信息，并可配准到所获取的三维结构信息中去。

Description

快速获取大样本三维结构信息和分子表型信息的成像装置和方法 [技术领域]
本发明涉及显微成像，具体地指一种快速获取大样本三维结构信息和分子表型信息的成像装置和方法。
[背景技术]
大脑是自然界最复杂的系统之一，支配着人类的一切活动。对大脑的探索一直是人类的研究目标，但时至今日我们仍无法准确描述智力、思维和意识的产生机制。神经结构是实现脑高级功能的重要基础。复杂的脑功能需要多个脑区的共同参与，并由局部和长程神经环路共同协同作用。脑疾病往往伴随着功能相关的特定神经环路及其输入连接与输出投射关系的结构异常。为了破译脑功能与脑疾病的结构基础，需要在全脑尺度进行细胞分辨水平地结构解析。此外，认识脑功能与脑疾病还需要解析其分子基础，明确参与环路与功能的神经元细胞类型，以便找出疾病发生、发展的分子机制。然而，神经元功能分子种类繁多，目前仅鉴定确认的类别就数以百计，这直接导致神经元分子表型纷繁复杂。以往仅按单一分子表型来进行神经元分类，不够准确。因此，找出特征分子表型以确定环路中神经元类型，需要在全脑尺度进行大量的筛选与鉴定，工作量巨大。
借助荧光标记和免疫组化技术，人们已经能够对脑区之间的神经连接进行示踪，对特定神经环路中细胞类型进行可视化。在相关研究中，通常需要先对特定神经环路进行荧光标记，再以传统组织学的方式，将完整大脑切成数百张薄片，逐片手工贴片和成像。在逐片检查了所有脑片的成像结果后，才能确定目标脑区，之后选择相应的脑片进行特定分子表型的免疫组化染色，最终鉴定出环路内神经元的细胞类型。上述传统方法均为手工操作，费时费力，效率低下，难以完成为基于分子表型进行细胞分类采集海量数据的工作。繁复的手工操作还导致一定数量的脑片存在耗损，无法获得全脑水平连续的神经投射信息。由于无法对相邻脑片空间位置进行配准，所获取的数据集无法重建三维结构。因此，神经结构的分子表型研 究急需发展自动化的全脑成像手段。
近年来，全脑免疫组化结合光片照明成像，提供了一种获取神经元分子表型的新工具，避免了大量的手工操作。利用透明后组织渗透性好的优点，已有光透明技术实现了全脑免疫组化染色，再通过光片照明成像技术对这些荧光标记的神经结构及其分子表型进行全脑成像。但受限于严重的技术缺陷，这些技术方案仅能提供全脑分布的简单参考，而难以展示神经环路纤维结构的分子表型。现有全脑免疫组化仅适用于小分子抗体，随着抗体分子量的增大，抗体的渗透深度与均匀性剧烈下降。全脑免疫组化的流程长且复杂，荧光信号易淬灭，样本存在变形，无法长期保存。抗体试剂使用量大，成本高。此外，目前光片照明技术成像分辨率较低，仅10μm左右，且在深部脑区进一步下降，无法在全脑范围获得一致的成像效果。成像所需超长工作距离物镜，设计难度高，价格昂贵。目前尚无可实现诸如全脑原位杂交等其他全脑分子表型染色的技术。
因此，要真正实现特定神经环路中神经元细胞类型的快速鉴定，亟待方法和技术上的突破。
[发明内容]
本发明目的在于克服上述现有技术的不足而提供一种快速获取大样本三维结构信息和分子表型信息的装置及方法，本发明解决了现有技术中样本制备流程复杂、影响样本形态和荧光信号、成像速度慢的缺点，能够快速获取并分析样本的三维结构信息，所获取数据具有自配准特性，并对感兴趣部位的样本切片进行分子表型染色，获得样本的分子表型信息，并可配准到所获取的三维结构信息中去。
实现本发明目的采用的技术方案是一种快速获取大样本三维结构信息和分子表型信息的成像装置，该装置包括：
样本存放装置；
三维移动台，用于驱动所述样本存放装置在三维空间内移动；
振动切片模块，用于对样本进行切片，得到样本的浅层部分；以及
宽场光学显微成像模块，用于对样本的浅层部分进行高通量层析成像。
此外，本发明还提供一种通过上述装置实现快速获取大样本三维结构信息和分子表型信息的方法，该方法包括：
步骤S101：利用荧光标记对生物组织样本进行标记；
步骤S102：使用琼脂糖包埋样本，得到包埋后的样本块；
步骤S103：将包埋后的样本块固定在水槽中，并加入缓冲液；
步骤S104：根据成像要求，在计算机中设置Z向采样间距、切片厚度、成像区间和Z向成像范围；
步骤S105：通过计算机控制三维平移台移动，使样本移至成像区域，并控制宽场光学显微成像模块对样本浅层整个断面进行高通量层析成像，并存储所得到的图像；
步骤S106：计算机控制三维平移台移动，使样本移至切片区域，并控制振动切片模块对样本已成像部分进行快速切片；
步骤S107：计算机控制切片收集模块将切片收集至容器中；
步骤S108：确认是否已完成样本整个Z向的成像，若没有完成则执行下一步骤；
步骤S109：计算机控制三维平移台沿Z向抬升样本，然后返回到步骤S105；若完成则执行下一步骤；
步骤S110：所获取的图像之间具有自配准性，可快速重建出样本三维结构信息成像数据并浏览，选取感兴趣部位的样本切片；
步骤S111：对选取的样本切片进行分子表型染色并成像，获取对应部位的分子表型信息，并配准至已有的三维结构信息成像数据。
本发明较现有技术具有以下优点：
(1)将振动切片与光学显微成像相结合，通过成像过程和切片过程的交替循环，克服了光学成像深度的限制，能够快速获取大样本的三维结构信息。与传统研究方法相比，提升了数据获取的效率且所获取的数据具有三维自配准特性，加速神经环路细胞分类的相关研究。
(2)在自动化的收集成像过程中产生的样本切片，能够根据样本三维结构信息的成像结果选择感兴趣的目标切片进行分子表型染色，获取样本的分子表型信息。避免了全脑免疫组化技术只能使用小分子抗体的局限性， 可适用于现有任意免疫组化抗体、原位杂交抗体等常用分子表型染色的试剂。
(3)采用琼脂糖包埋样本，流程简单，不会造成样本形变，不影响样本的荧光信号和抗原特性。
[附图说明]
图1为本发明快速获取大样本三维结构信息和分子表型信息成像装置的结构示意图。
图2为本发明中计算机的控制连接框图。
图3为本发明快速获取大样本三维结构信息和分子表型信息成像方法的流程图。
图4为本发明实现快速获取大样本三维结构信息的示意图。
图5为本发明中获取的鼠脑样本的三维结构数据图，图5a为全脑结构数据的三维重建结果，图5b为单个冠状面图。
图6为本发明中获取的样本切片的免疫组化数据图，图6a为抗-小清蛋白的免疫组化图；图6b为抗-钙结合蛋白的免疫组化图。
[具体实施方式]
下面结合附图和具体实施例对本发明作进一步的详细说明。
本实施例快速获取大样本三维结构信息和分子表型信息成像装置的结构如图1所示，该装置包括精密三维平移台9、水槽7、宽场光学显微成像模块、振动切片模块、切片收集模块和计算机15。水槽7设于精密三维平移台9上，水槽7内装有缓冲液，样本5置于缓冲液中。精密三维平移台9与计算机15连接，计算机15控制精密三维平移台9在三维方向移动。
本实施例所用宽场光学显微成像模块为结构光照明显微镜，用于对样本的浅层部分进行高通量层析成像，它包括光源1、空间光调制器2、物镜3和相机4，空间光调制器2用于调制所述光源发出的光，物镜3用于对空间光调制器调制后的光形成结构光调制条纹，即由光源1出射的光经空间 光调制器2调制后在物镜3的焦面上形成结构光调制条纹。相机4用于获取不同相位的图像后传输至计算机15。将三幅不同相位的图像经过结构光照明成像重建算法计算后即可得到样本的层析图像。
本实施例中所用振动切片模块8用于将样本的已成像部分切除，振动切片模块8为现有常用设备，此处不再赘述。振动切片模块8的刀片6位于水槽7内缓冲液面以下。
本实施例中所用切片收集模块用于收集切除得到的切片，它包括容器12、管路10和水泵11，管路的一端对着刀片6处(切片被切除的部位)，另一端通向容器12；水泵11设于管路10中，用于将水槽7内的缓冲液抽入到容器12内。
如图2所示，本实施例中计算机15分别与空间光调制器2、相机4、振动切片模块8、精密三维平移台9和水泵11连接。计算机15对上述各部件的控制分别为：计算机15通过控制空间光调制器2和相机4实现结构光显微成像，获取样本的层析图像并存储在计算机15中；计算机15通过控制振动切片模块8和精密三维平移台9实现对样本5的快速切片；计算机15通过控制水泵11实现将水槽7中的缓冲液抽入到容器12内，被切除的切片随缓冲液也被抽入到容器12内。
本实施例中所用各部件的参数具体为：光源1采用Lumen Dynamics公司生产的X-cite exact金属卤化物光源；空间光调制器2采用规格为0.7XGA的数字微镜阵列；成像用的物镜3为日本Olympus公司生产的NA值为1.0的20×消色差物镜；成像相机4为日本Hamamatsu公司生产的sCMOS相机，像素规格为2048×2048；水槽7为金属加工件；采用基于弹簧钢片结构和电磁力驱动的振动切片模块8，刀片6采用美国Electron Microscopy Sciences公司的氧化锆刀片；精密三维平移台9采用美国Aerotech公司的产品，固定于大理石平台上，定位精度为亚微米水平，能满足切片和成像的精度要求；切片收集模块中的水泵11为高流量隔膜泵。
通过本实施例快速获取大样本三维结构信息和分子表型信息的方法流程如图3所示，具体包括以下步骤：
步骤S101：利用荧光标记技术对生物组织样本的特定结构进行标记；
步骤S102：使用琼脂糖包埋样本，得到包埋后的样本块。本实施例将新鲜的样本组织经过固定后，采用3％～5％的琼脂糖包埋，包埋过程仅需1～2个小时。
步骤S103：将包埋后的样本固定在水槽中7，并加入硼酸钠缓冲液。
步骤S104：根据成像要求，在计算机15上设置Z向采样间距、切片厚度、成像区间和Z向成像范围；
步骤S105：计算机控制精密三维平移台9将样本移至成像区域，并控制结构光照明显微镜对样本5的浅层部分13进行高通量层析成像，相机4获取不同相位的图像后由计算机通过重建算法计算得到样本的层析图像；并存储所得到的图像；
步骤S106：计算机控制精密三维平移台9将样本移至切片区域，并控制振动切片模块对样本浅层已成像部分13进行快速切片；
步骤S107：计算机控制切片收集模块将切片收集至指定容器12中；
步骤S108：确认是否已完成样本整个Z向的成像，若没有完成则执行下一步骤；
步骤S109：Z向抬升样本，返回到步骤S105，继续对暴露出的样本浅层部分14进行成像和切片；若完成则执行下一步骤；
步骤S110：所获取的图像之间具有自配准性，可快速重建出样本三维结构信息成像数据并浏览，选取感兴趣部位的样本切片；
步骤S111：对选取的样本切片进行分子表型染色并成像，获取对应部位的分子表型信息，并配准至已有的三维结构信息成像数据。
如图4所示，通过上述方法实现快速获取大样本三维结构信息的过程为：宽场光学显微成像模块通过物镜3对样本5的浅层部分13进行高通量层析成像，成像完成后振动切片模块8将样本已成像部分13切除，切片收集模块收集切片，随后计算机15控制精密三维平移台9沿Z向抬升样本，继续对样本浅层部分14进行成像，成像完成后将浅层部分14切除，切片、收集和成像过程循环即可获取整个样本的间隔采样层析图像和切片。
图5为采用上述快速获取大样本三维结构信息和分子表型信息的成像方法获取的鼠脑样本的三维结构数据图。利用转基因荧光标记技术对该鼠脑进行标记，该鼠脑中含有促肾上腺皮质激素释放激素基因的细胞均表达荧光蛋白，其中，图5a为全脑结构数据的三维重建结果图，图5b为单个冠状面图片。整套数据包含300层图像，每层图像之间间隔50μm。图像的分辨率达到了0.32μm×0.32μm，能够清晰的分辨胞体和纤维等细节信息。图像之间具备自配准特性，能够方便的得到三维重建结果，并辨别含有促肾上腺皮质激素释放激素基因的细胞在全脑范围内的分布和聚集情况。整个全脑结构数据的采集时间仅需12小时。
图6为采用上述快速获取大样本三维结构信息和分子表型信息的成像方法获取的鼠脑样本皮层部位切片的免疫组化数据图，其中，图6a为抗-小清蛋白的免疫组化图片，图6b为抗-钙结合蛋白的免疫组化图片。根据已有的三维结构数据结果可知皮层部位有较多的含有促肾上腺皮质激素释放激素基因的细胞，故选着含皮层的样本切片进行免疫组化染色。染色操作为常规的操作步骤，简单、快速，可适用于现有任意免疫组化抗体、原位杂交抗体等常用分子表型染色的试剂。图中免疫组化数据显示了鼠脑皮层部位包含小清蛋白和钙结合蛋白的细胞分布情况。
尽管结合优选实施方案具体展示和介绍了本发明，但所属领域的技术人员应该明白，在不脱离所附权利要求书所限定的本发明的精神和范围内，在形式上和细节上可以对本发明做出各种变化，均为本发明的保护范围。

Claims (7)

  1. 一种快速获取大样本三维结构信息和分子表型信息的成像装置，其特征在于，包括：
    样本存放装置；
    三维移动台，用于驱动所述样本存放装置在三维空间内移动；
    振动切片模块，用于对样本进行切片，得到样本的浅层部分；以及
    宽场光学显微成像模块，用于对样本的浅层部分进行高通量层析成像。
  2. 根据权利要求1所述快速获取大样本三维结构信息和分子表型信息的成像装置，其特征在于，还包括：
    切片收集模块，用于收集切除得到的切片。
  3. 根据权利要求2所述快速获取大样本三维结构信息和分子表型信息的成像装置，其特征在于所述切片收集模块包括：
    容器；
    管路，其一端对着切片被切除的部位，另一端通向所述容器；以及
    水泵，设于管路中，用于将被切除的切片随缓冲液从管路中抽入到所述容器内。
  4. 根据权利要求3所述快速获取大样本三维结构信息和分子表型信息的成像装置，其特征在于，还包括：
    计算机，分别与所述三维平移台、宽场光学显微成像模块、振动切片模块和切片收集模块连接，用于控制所述三维平移台、宽场光学显微成像模块、振动切片模块和切片收集模块实现各自的工作。
  5. 根据权利要求1～4任一项所述快速获取大样本三维结构信息和分子表型信息的成像装置，其特征在于：所述样本存放装置内装有缓冲液，样本置于缓冲液中。
  6. 根据权利要求5所述快速获取大样本三维结构信息和分子表型信息的成像装置，其特征在于：所述宽场光学显微成像模块为结构光照明显微镜或其他能实现高通量层析成像的光学仪器。
  7. 一种通过权利要求1所述装置实现快速获取大样本三维结构信息和分子表型信息的方法，其特征在于，包括：
    步骤S101：利用荧光标记对生物组织样本进行标记；
    步骤S102：使用琼脂糖包埋样本，得到包埋后的样本块；
    步骤S103：将包埋后的样本块固定在水槽中，并加入缓冲液；
    步骤S104：根据成像要求，在计算机中设置Z向采样间距、切片厚度、成像区间和Z向成像范围；
    步骤S105：通过计算机控制三维平移台移动，使样本移至成像区域，并控制宽场光学显微成像模块对样本浅层整个断面进行高通量层析成像，并存储所得到的图像；
    步骤S106：计算机控制三维平移台移动，使样本移至切片区域，并控制振动切片模块对样本已成像部分进行快速切片；
    步骤S107：计算机控制切片收集模块将切片收集至容器中；
    步骤S108：确认是否已完成样本整个Z向的成像，若没有完成则执行下一步骤；
    步骤S109：计算机控制三维平移台沿Z向抬升样本，然后返回到步骤S105；若完成则执行下一步骤；
    步骤S110：所获取的图像之间具有自配准性，快速重建出样本三维结构信息成像数据并浏览，选取感兴趣部位的样本切片；
    步骤S111：对选取的样本切片进行分子表型染色并成像，获取对应部位的分子表型信息，并配准至已有的三维结构信息成像数据。
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