CN111999329A - A sample stage for cryo-EM imaging - Google Patents
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- CN111999329A CN111999329A CN202010883086.2A CN202010883086A CN111999329A CN 111999329 A CN111999329 A CN 111999329A CN 202010883086 A CN202010883086 A CN 202010883086A CN 111999329 A CN111999329 A CN 111999329A
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Abstract
本发明公开了一种用于冷冻电镜成像用样品台,包括底座和可拆卸的填充部件,所述底座为圆盘状结构,所述底座上设有凸沿,所述凸沿与底座围成用于容置样品的空腔,所述填充部件能够在高压冷冻时对所述凸沿的外侧壁与底座形成的阶梯结构进行填充,并在预减薄过程中取下。本发明可以容纳加载高端冷冻透射电镜上样环,并可稳定地被固定在上样环和上样底座之间,并可以被电镜Autoloader中的机械手抓取,并放入镜筒中进行成像。因此,本发明提供的样品台可应用于Autoloader的冷冻电镜上。
The invention discloses a sample stage for cryo-electron microscope imaging, comprising a base and a detachable filling part, the base is a disc-shaped structure, and a convex edge is arranged on the base, and the convex edge and the base are enclosed The cavity for accommodating the sample, the filling part can fill the stepped structure formed by the outer side wall of the flange and the base during high-pressure freezing, and can be removed during the pre-thinning process. The invention can accommodate and load a high-end frozen transmission electron microscope sample loading ring, can be stably fixed between the sample loading ring and the sample loading base, can be grasped by a manipulator in the electron microscope Autoloader, and put into a lens barrel for imaging. Therefore, the sample stage provided by the present invention can be applied to the cryo-electron microscope of Autoloader.
Description
技术领域technical field
本发明涉及生物样品检测领域,特别涉及一种用于冷冻电镜成像用样品台。The invention relates to the field of biological sample detection, in particular to a sample stage for cryo-electron microscope imaging.
背景技术Background technique
近年来,得益于冷冻电子断层成像技术(cryo-electron tomography,cryo-ET)的发展,原位结构生物学研究发展迅速,其研究对象的状态更接近生理条件,解析结构的分辨率也在不断提高。其最终目的是原位解析生物大分子及分子机器的结构,从而在分子、细胞器、细胞等不同层次上阐述生命活动的本质。In recent years, thanks to the development of cryo-electron tomography (cryo-ET), in situ structural biology research has developed rapidly. keep improving. Its ultimate purpose is to analyze the structure of biological macromolecules and molecular machines in situ, so as to explain the essence of life activities at different levels such as molecules, organelles, and cells.
冷冻电子断层成像技术一般要求样品厚度在500nm以下,否则成像时会产生过多的非弹性散射电子使得照片信噪比严重下降,后续图像处理难以进行。由于绝大部分细胞和组织样品的厚度远超500nm,因此这类样品采取的技术路线是冷冻固定——冷冻聚焦离子束(Cryo-FIB)减薄——冷冻电子断层成像。通常,一些小细胞通过快速冷冻的方式,冷冻固定于电镜专用的载网之上,然后通过冷冻聚焦离子束减薄后进行冷冻电镜成像。但是快速冷冻的冷冻深度最多在十几个微米,对厚度动辄在几十到几百微米的组织和一些大尺度细胞而言,单纯的快速冷冻无法使样品结构得到良好的固定,因此必须通过高压冷冻的技术。高压冷冻后产生的样品块经过冷冻切片机的修块,再通过冷冻聚焦离子束减薄到几百纳米,最终送入冷冻电镜进行成像。Cryo-electron tomography generally requires the thickness of the sample to be less than 500 nm, otherwise too many inelastic scattered electrons will be generated during imaging, which will seriously reduce the signal-to-noise ratio of the photo and make subsequent image processing difficult. Since the thickness of the vast majority of cell and tissue samples is well above 500 nm, the technical route taken for such samples is cryo-fixation - cryo-focused ion beam (Cryo-FIB) thinning - cryo-electron tomography. Usually, some small cells are frozen and fixed on a special carrier grid for electron microscope by means of rapid freezing, and then cryo-EM imaging is performed after being thinned by a frozen focused ion beam. However, the freezing depth of rapid freezing is at most a dozen microns. For tissues and some large-scale cells with a thickness of tens to hundreds of microns, simple rapid freezing cannot fix the sample structure well, so high pressure must be used. Freezing technology. The sample block produced after high-pressure freezing is trimmed by a cryostat, and then thinned to several hundreds of nanometers by a frozen focused ion beam, and finally sent to a cryo-electron microscope for imaging.
为了提高成像分辨率,人们使用带有Autoloader的冷冻电镜,如FEI Titan Kros进行成像,这类电镜分辨率高,可以得到更加真实清晰的原位结构。然而,电镜所用的样品承载工具是载网,高压冷冻所用的样品承载工具是样品台,Autoloader无法装载样品台,因此,现有的样品台无法应用到Autoloader的冷冻电镜上。In order to improve the imaging resolution, people use cryo-electron microscopes with Autoloader, such as FEI Titan Kros, for imaging, which have high resolution and can obtain more real and clear in-situ structures. However, the sample carrier used in the electron microscope is the carrier mesh, and the sample carrier used in the high-pressure freezing is the sample stage. The Autoloader cannot load the sample stage. Therefore, the existing sample stage cannot be applied to the Autoloader's cryo-EM.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于提供一种用于冷冻电镜成像用样品台,以解决现有的样品台无法应用到Autoloader的冷冻电镜上的问题。The purpose of the present invention is to provide a sample stage for cryo-electron microscope imaging, so as to solve the problem that the existing sample stage cannot be applied to the cryo-electron microscope of Autoloader.
根据本发明的实施例,提供了一种用于冷冻电镜成像用样品台,包括底座和可拆卸的填充部件,所述底座为圆盘状结构,所述底座上设有凸沿,所述凸沿与底座围成用于容置样品的空腔,所述填充部件能够在高压冷冻时对所述凸沿的外侧壁与底座形成的阶梯结构进行填充,并在预减薄过程中取下。According to an embodiment of the present invention, a sample stage for cryo-electron microscopy imaging is provided, comprising a base and a detachable filling part, the base is a disc-shaped structure, a convex edge is provided on the base, and the convex The edge and the base form a cavity for accommodating the sample, and the filling part can fill the stepped structure formed by the outer side wall of the flange and the base during high-pressure freezing, and can be removed during the pre-thinning process.
具体地,所述凸沿和填充部件均为环状结构,所述填充部件能够套设在所述凸沿的外侧壁上。Specifically, the protruding edge and the filling member are both annular structures, and the filling member can be sleeved on the outer side wall of the protruding edge.
具体地,所述凸沿的中心与所述底座的中心相重合。Specifically, the center of the protruding edge coincides with the center of the base.
具体地,所述填充部件的高度与所述凸沿外侧壁的高度相同。Specifically, the height of the filling member is the same as the height of the outer side wall of the protruding edge.
具体地,所述底座和填充部件采用导热的金属材料制成。Specifically, the base and the filling part are made of thermally conductive metal materials.
本发明实施例提供了一种用于冷冻电镜成像用样品台,可以容纳加载高端冷冻透射电镜上样环,并可稳定地被固定在上样环和上样底座之间,并可以被电镜Autoloader中的机械手抓取,并放入镜筒中进行成像。因此,本发明提供的样品台可应用于Autoloader的冷冻电镜上。The embodiment of the present invention provides a sample stage for cryo-EM imaging, which can accommodate and load a high-end cryo-TEM sample loading ring, can be stably fixed between the sample loading ring and the sample loading base, and can be loaded by the electron microscope Autoloader The manipulator grabs it and puts it into the lens barrel for imaging. Therefore, the sample stage provided by the present invention can be applied to the cryo-electron microscope of Autoloader.
附图说明Description of drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.
图1为本发明实施例提供的一种冷冻电镜成像用样品台的结构图;1 is a structural diagram of a sample stage for cryo-EM imaging provided by an embodiment of the present invention;
图2为底座的结构图;Fig. 2 is the structure diagram of the base;
图3为填充部件的结构图;Fig. 3 is the structure diagram of filling part;
图4为本发明实施例提供的一种冷冻电镜成像用样品台的使用状态图。FIG. 4 is a use state diagram of a sample stage for cryo-EM imaging according to an embodiment of the present invention.
其中,1-底座,2-填充部件,3-凸沿,4-空腔,5-上样底座,6-上样环。Among them, 1- base, 2- filling part, 3- convex edge, 4- cavity, 5- sample loading base, 6- sample loading ring.
具体实施方式Detailed ways
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整的描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.
根据本发明的实施例,参见图1至图3,提供了一种用于冷冻电镜成像用样品台,包括底座1和可拆卸的填充部件2,底座1为圆盘状结构,底座1上设有凸沿3,凸沿3与底座1围成用于容置样品的空腔4,填充部件2能够在高压冷冻时对凸沿3的外侧壁与底座1形成的阶梯结构进行填充,并在预减薄过程中取下。According to an embodiment of the present invention, referring to FIGS. 1 to 3 , a sample stage for cryo-electron microscopy imaging is provided, comprising a
其中,凸沿3和填充部件2均为环状结构,填充部件2能够套设在凸沿3的外侧壁上,方便工作人员安装和取下填充部件2。凸沿3的中心与底座1的中心相重合。填充部件2的高度与凸沿3外侧壁的高度相同。底座1和填充部件2采用导热的金属材料制成,导热的金属材料可为采用纯铜/银/铜镀金等,但不限于上述导热材料,本实施例不做限定。样品台的尺寸可由实际需求设置,本实施例不做限定。The protruding edge 3 and the filling part 2 are both annular structures, and the filling part 2 can be sleeved on the outer side wall of the protruding edge 3 to facilitate the staff to install and remove the filling part 2 . The center of the convex edge 3 coincides with the center of the
样品切片的制作过程为:首先,在高压冷冻部分,将填充部件2套设在凸沿3的外侧壁上,以使填充部件2将凸沿3的外侧壁与底座1形成的阶梯结构进行填充,将要冷冻的生物样品放置于空腔4中,加入冷冻保护剂填充空腔4,然后在其上覆盖蓝宝石片,组成一个整体,放入高压冷冻仪中进行冷冻固定。填充部件2填充了蓝宝石片与底座1之间形成的空间,提高冷冻效果及冷冻速度。The production process of the sample slice is as follows: first, in the high-pressure freezing part, the filling part 2 is sleeved on the outer side wall of the convex edge 3, so that the filling part 2 fills the stepped structure formed by the outer side wall of the convex edge 3 and the
其次,冷冻后的整体在液氮中转移至冷冻超薄切片机。取下蓝宝石片后将底座1与套环的组合体进行预减薄,修剪掉约三分之一至二分之一,即由圆形修剪至半圆形/大半圆形,暴露出冷冻后的生物样品表面,将其由约百微米尺度减薄至几十微米尺度。修剪为半圆形后,填充部件2可以轻易取下,不影响样品台的后续使用。本步骤的目的是暴露样品、取下套环,便于后续FIB切割和电镜成像。Second, the frozen whole was transferred to a cryomicrotome in liquid nitrogen. After removing the sapphire sheet, pre-thin the combination of the
然后,如图4所示,为预减薄后的样品台的底座1加载上样环6(c-clip)和上样底座5(AutoGrid),样品台被稳定的固定在二者之间,形成上样环6-样品台-上样底座5整体,并在液氮中转移至扫描电镜冷冻腔室,进行冷冻聚焦离子束(cryo-FIB)减薄。减薄后的样品厚度在200nm左右,可以满足透射电镜成像的要求。Then, as shown in Figure 4, a sample loading ring 6 (c-clip) and a loading base 5 (AutoGrid) are loaded on the
最后,上样环6-样品台-上样底座5整体被电镜Autoloader中的机械手抓取,并放入镜筒中,进行冷冻透射电镜成像及数据收集。Finally, the sample loading ring 6-sample stage-
本发明实施例提供了一种用于冷冻电镜成像用样品台,可以容纳加载高端冷冻透射电镜上样环6,并可稳定地被固定在上样环6和上样底座5之间,并可以被电镜Autoloader中的机械手抓取,并放入镜筒中进行成像。因此,本发明提供的样品台可应用于Autoloader的冷冻电镜上。The embodiment of the present invention provides a sample stage for cryo-EM imaging, which can accommodate the loading of a high-end cryo-TEM sample loading ring 6, and can be stably fixed between the sample loading ring 6 and the
本领域技术人员在考虑说明书及实践这里公开的发明后,将容易想到本发明的其它实施方案。本申请旨在涵盖本发明的任何变型、用途或者适应性变化,这些变型、用途或者适应性变化遵循本发明的一般性原理并包括本发明未公开的本技术领域中的公知常识或惯用技术手段。说明书和实施例仅被视为示例性的,本发明的真正范围和精神由下面的权利要求指出。Other embodiments of the invention will readily suggest themselves to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses or adaptations of the invention which follow the general principles of the invention and which include common knowledge or conventional techniques in the art not disclosed by the invention . The specification and examples are to be regarded as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.
应当理解的是,本发明并不局限于上面已经描述并在附图中示出的精确结构,并且可以在不脱离其范围进行各种修改和改变。本发明的范围仅由所附的权利要求来限制。It should be understood that the present invention is not limited to the precise structures described above and illustrated in the accompanying drawings, and that various modifications and changes may be made without departing from the scope thereof. The scope of the present invention is limited only by the appended claims.
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CN113252717A (en) * | 2021-06-15 | 2021-08-13 | 中国科学院地质与地球物理研究所 | Intelligent preparation system and method for frozen electron microscope sample and electronic equipment |
CN113252717B (en) * | 2021-06-15 | 2021-09-10 | 中国科学院地质与地球物理研究所 | Intelligent preparation system and method for frozen electron microscope sample and electronic equipment |
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