CN114563432A - 一种用于扫描电子显微镜三维结构重建实验的冷冻样品处理方法 - Google Patents
一种用于扫描电子显微镜三维结构重建实验的冷冻样品处理方法 Download PDFInfo
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Abstract
本发明涉及实验样品处理相关技术领域，公开了一种用于扫描电子显微镜三维结构重建实验的冷冻样品处理方法，包括如下步骤：S1:取样，将样品内空气排出，利用冷冻保护剂填充后将样品冷冻固定；S2：将所述步骤S1中取得的样品放置于冷冻替代液中进行冷冻替代程序；S3：对所述步骤S2中取得的样品进行漂洗和复水S4：利用醛类物质对所述步骤S3中取得的样品进行漂洗及固定，可保存生物样品内部近似于自然状态下的超微结构信息。可制备用于扫描电镜三维结构重建实验的大尺寸样品。所制备的样品在扫描电镜钟背散射电子图像反差高，样品的生物膜结构清晰可见。所制备的样品可与透射电镜成像系统联合使用，无需切片后做重金属染色。
Description
技术领域
本发明涉及实验样品处理相关技术领域，具体为一种用于扫描电子显微镜三维结构重建实验的冷冻样品处理方法。
背景技术
生命体微观组织结构反映其生理活动，因此，研究人员聚焦于观察生命体在细胞、组织、器官甚至完整个体水平上的形貌与行为，以探究生命体的微观结构与功能的关系。为了满足这一研究需求，扫描电子显微镜(Scanning Electron Microscope，简称SEM)得到了广泛地应用。凭借这一强有力的科研工具，研究人员可以在微观、纳观尺度下对样品外部形貌进行表征、测试等一系列操作，这对于理解生命体微观结构的作用机理、指导临床病理研究、生物病虫害防治等都具有重要意义。在一系列利用SEM进行的科学研究中，样品横切面的扫描电镜背散射电子成像(Serial blockface scanning electron microscopy，SBEM)实验受到广泛关注。它是通过对生物样品进行脱水处理和树脂包埋以方便后期对样品均匀切割，实现对生物样品平整的横切面进行背散射电子成像，并且通过连续的样品切割和横截面成像实现样品内部结构的三维数据收集，再经过后期的计算机图像处理最终获取样品在三维空间中的内部结构信息。这克服了传统二维电镜技术中只能获取样品某个切面的结构信息而产生的结构信息局限性，极大的满足了生科科学研究的需要。目前，部分商业化的扫描电子显微镜具有对样品进行三维数据收集的功能，现有的生物电镜样品制备方法主要是通过对样品进行醛类物质的前固定和锇酸的后固定处理以达到保存样品微观结构的目的，后期再经过树脂包埋、超薄切片和重金属铅和铀双染色等处理，最后进行电镜观察。以上的样品制备方法是目前国内外使用最广泛的一种方法，在揭示生命体微观结构研究中发挥了十分重要的作用。然而就现有的生物样品制备方法来说还存在一些弊端，主要包括醛类物质对生物样本固定速度慢导致微观结构改变，一步锇酸固定法无法提供足够的背散射信号导致图像反差小进而影响分辨率，此外生物体主要是碳、氢、氧、磷、硫等轻元素构成，在扫描电镜下的背散射电子信号产额很低需经过重金属染色来提高信号量，而现有方法是对样品进行切片后再用重金属染色，这与SBEM成像方法无法兼容等。基于此，开发一种既可以快速固定样品结构信息又能够在扫描电镜中进行高分辨率的背散射电子成像的新型样品制备方法十分必要，针对上述问题，提出了本申请。
发明内容
本发明的目的在于提供一种用于扫描电子显微镜三维结构重建实验的冷冻样品处理方法，用于解决上述问题。
本发明是通过以下技术方案来实现的。
本发明的生物样品制备方法，包括如下步骤：
S1:取样，将样品内空气排出，利用冷冻保护剂填充后将样品快速冷冻固定；
S2：对样品放置于冷冻替代液中进行冷冻替代程序；
S3：对样品进行漂洗和复水
S4：利用醛类物质对样品进行漂洗及固定；
S5：利用两步锇酸固定法处理样品，满足样品在电镜下的被散射电子信号成像；
S6：对样品进行铀染，铀染后漂洗；
S7：对样品进行铅染，铅染后漂洗；
S8：将样品进行脱水处理，利用包埋剂浸透样品；
S9：将样品放入包埋板上盛有包埋剂的包埋孔中，进行整姿处理，将包埋板加热使包埋剂聚合，样品处理完成。
进一步地，所述步骤S1中的冷冻保护剂为十六碳烯。
进一步地，所述步骤S1中，样品被抽气处理时的气压为0.09MPa，持续时间为5min。
进一步地，所述步骤S1中，在样品被快速冷冻之前，去除样品表面多余的十六碳烯。
进一步地，所述步骤S2中，冷冻替代液为用纯丙酮、醋酸双氧铀和戊二醛配制的溶液。
进一步地，所述步骤S2中，冷冻替代程序为：将温度从-90℃逐渐升温到0℃。
进一步地，所述S4步骤的具体步骤如下：
S41:将装有样品的容器转移到0摄氏度的载台上，并利用预冷的含有戊二醛的丙酮溶液漂洗两次；
S42:用梯度丙酮溶液对漂洗好的样品进行复水处理，依次在每个梯度溶液中浸泡；
S43:将样品分别在固定液I和固定液II中浸泡；
S44:将样品在HEPES缓冲液浸泡；
S45:将样品在PBS缓冲液浸泡。
进一步地，所述步骤S5的具体步骤如下：
S51:将样品转移到锇酸固定液A中，并置于0摄氏度的载台上，然后用双蒸水漂洗；
S52:将样品转移至硫代对称二氨基脲中孵育，然后用双蒸水漂洗；
S53:将样品转移至锇酸中，然后漂洗。
进一步地，所述步骤S6的具体步骤如下：
S61：将样品转移至醋酸双氧铀中染色，然后漂洗。
进一步地，所述步骤S7的具体步骤如下：
S71：将样品转移至铅染液中孵育，然后漂洗。
进一步地，所述步骤S71中，铅染液的配制：将天冬氨酸溶解于水中配制成天冬氨酸缓冲液，将硝酸铅溶解于天冬氨酸缓冲液中，调节pH值到5.5。
进一步地，所述步骤S41中，所述梯度丙酮溶液为用水和纯丙酮配制的浓度在95％～70％之间的若干个浓度的溶液，且每个浓度的溶液中均含有戊二醛。
本发明的有益效果：
可保存生物样品内部近似于自然状态下的超微结构信息。
可制备用于扫描电镜三维结构重建实验的大尺寸样品，尺寸最大可达8mm3。
所制备的样品在扫描电镜钟背散射电子图像反差高，样品的生物膜结构清晰可见。
所制备的样品可与透射电镜成像系统联合使用，无需切片后做重金属染色。
使用两步锇酸固定法处理样品，满足样品在电镜下的被散射电子信号成像。
具体实施方式
下面通过实施例对本发明进行详细说明。
冷冻替代液的配制：称取0.1g醋酸乙酸双氧铀固体粉末溶于1ml甲醇中配成10％乙酸双氧铀的甲醇溶液备用。取0.1ml的10％乙酸双氧铀、0.1ml的20％戊二醛的丙酮溶液和9.8ml的纯丙酮混匀，经搅拌混合制得，分装在1.5ml的离心管中，每管加1.3ml冷冻替代液，并在-196℃条件下保存。
1％(质量分数)硫代对称二氨基脲的配制：称取0.1g硫代对称二氨基脲固体粉末，溶解于10ml的去离子水里，置于60℃烘箱中，每过5min上下颠倒混匀直至硫代对称二氨基脲固体彻底溶解，待溶液温度恢复至室温，利用水系的0.22μm孔径的滤膜过滤制得，试剂室温保存备用，需现用现配。
铅染液的配制：称取0.0998g天冬氨酸溶解于25ml水中配制成天冬氨酸缓冲液，期间需把缓冲液置于60℃烘箱中加入，并每过5min上下颠倒直至彻底溶解。称取0.165g硝酸铅溶解于25ml的天冬氨酸缓冲液中，使用1M(1mol/L)的氢氧化钠溶液调节PH值到5.5，最后置于室温备用。
包埋剂的配制：将EMBED-812-RESIN、DDSA、NMA和DMP-30，逐样加入烧杯中，用铝箔纸密封烧杯口，用磁力搅拌器搅动均匀，搅动约1小时，去掉铝箔纸，用真空干燥器将包埋剂中的气泡抽出，并在干燥器中静置片刻待气泡完全消失备用，其中EMBED-812-RESIN为环氧树脂，DDSA为2-十二烯基丁二酸酐，NMA为甲基纳迪克酸酐，DMP-30为2-4-6-三(二甲氨基甲基)苯酚。
一种用于扫描电子显微镜三维结构重建实验的冷冻样品处理方法，依次由下述步骤组成：
(1)取新鲜的昆虫样品用手术刀在提前用PBS湿润的滤纸上把虫体切成长、宽和高均不超过2mm的组织块，并用镊子轻轻夹住迅速浸入到装有冷冻保护剂的1.5ml离心管中。
(2)将装有样品的离心管放入真空箱中进行抽气处理，真空箱内压力控制在0.09Mpa，处理时间为5min，使得样品内部的空气排出并被冷冻保护剂填充。
(3)用镊子从冷冻保护剂中夹住组织块在滤纸上轻轻蘸一下，吸取组织块表面多余的冷冻保护剂，然后快速将组织块投入液态乙烷中对样品进行快速冷冻固定。
(4)预先准备好冷冻替代液并冷冻在液氮中，利用镊子将样品快速转移至液氮中，然后在液氮下把样品转移到装有冷冻替代液的管子中并盖好盖子。
(5)预先将冷冻替代仪加满液氮预冷，使样品仓温度维持在-90℃，将装有样品和冷冻替代液的管子迅速转移到冷冻替代仪的样品仓中，并开始冷冻替代程序，冷冻替代程序为：-90℃中持续72小时，再以每小时2℃的速度升温到-60℃，-60℃中持续20小时，再以每小时2℃的速度升温到-30℃，-30℃中持续15小时，再以每小时5℃的速度升温到0℃。
(6)待冷冻替代程序结束，把装有样品的管子转移到温度低于0°的载体上，本实施例中载体为冰，并利用预冷的含有0.4％(质量分数)戊二醛的丙酮溶液漂洗两次，每次30min。
(7)用水和纯丙酮配制95％、90％、80％、70％梯度丙酮溶液，且每个浓度的丙酮中均含有0.5％，将样品在以上梯度丙酮溶液中进行复水处理，依次在每个梯度溶液中浸泡20min，所有操作在冰上进行。
(8)将样品在含有0.5％(质量分数)戊二醛的50％丙酮溶液中浸泡20min，再将样品转移到含有0.5％(质量分数)戊二醛的30％丙酮溶液中浸泡20min。
(9)将样品在0.1mol/L HEPES(4-羟乙基哌嗪乙磺酸)缓冲液浸泡20min。
(10)将样品在0.1mol/L PBS(PH 7.4)缓冲液(磷酸缓冲盐溶液)浸泡20min。
(11)将样品转移到锇酸固定液A中，并置于冰上，1小时后用双蒸水漂洗3次，每次10min。其中锇酸固定液A是用2％(质量分数)锇酸和3％(质量分数)的亚铁氰化钾水溶液等体积比例混合配制而成。
(12)将样品转移至室温状态下的1％(质量分数)硫代对称二氨基脲中孵育20min，然后用双蒸水漂洗3次，每次10min。
(13)将样品转移至2％(质量分数)锇酸中，室温固定30min后，用双蒸水漂洗3次，每次10min。
(14)将样品转移至1％(质量分数)醋酸双氧铀中在4℃下染色12小时，然后用双蒸水漂洗3次，每次10min。
(15)将样品转移铅染液中，在60℃下孵育30min，然后用双蒸水漂洗3次，每次10min。
(16)用30％、50％、70％、80％和90％梯度乙醇溶液及丙酮对漂洗好的样品进行脱水处理，每次处理30min。再用100％的乙醇处理20min，用纯丙酮对样品进行过渡处理3次，每次15min，再用体积比为3:7和7:3的丙酮和包埋剂的混合液对样品进行梯度浸透，依次处理样品8小时和15小时，然后换管，用纯包埋剂处理样品2次，每次24小时。
(17)EMBED-812-RESIN、DDSA、NMA和DMP-30按体积份数分别加入18.8份、9.9份、11.3份和0.6份，充分混匀后备用。将样品用牙签从纯包埋剂中挑出置于滤纸上，吸掉样品表面的包埋剂，然后放入包埋板上盛有包埋剂的包埋孔中，再进行整姿处理，将包埋板放入烘箱中加热使包埋剂聚合，样品处理完成。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此领域技术的人士能够了解本发明内容并加以实施，并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围内。
Claims (12)
1.生物样品制备方法，其特征在于：包括如下步骤：
S1:取样，将样品内空气排出，利用冷冻保护剂填充后将样品冷冻固定；
S2：将所述步骤S1中取得的样品放置于冷冻替代液中进行冷冻替代程序；
S3：对所述步骤S2中取得的样品进行漂洗和复水
S4：利用醛类物质对所述步骤S3中取得的样品进行漂洗及固定；
S5：利用两步锇酸固定法处理所述步骤S4中得到样品；
S6：对样品进行铀染，铀染后漂洗；
S7：对样品进行铅染，铅染后漂洗；
S8：将样品进行脱水处理，利用包埋剂浸透样品；
S9：将利用包埋剂浸透的样品放入包埋板上盛有包埋剂的包埋孔中，进行整姿处理，将包埋板加热使包埋剂聚合，样品处理完成。
2.根据权利要求1所述的生物样品制备方法，其特征在于：所述步骤S1中的冷冻保护剂为十六碳烯。
3.根据权利要求1或2所述的生物样品制备方法，其特征在于：所述步骤S1中，样品被抽气处理时的气压为0.09MPa。
4.根据权利要求2所述的生物样品制备方法，其特征在于：所述步骤S1中，在样品被快速冷冻之前，去除样品表面多余的十六碳烯。
5.根据权利要求1、2、4所述的生物样品制备方法，其特征在于：所述步骤S2中，冷冻替代液为用纯丙酮、醋酸双氧铀和戊二醛配制的溶液。
6.根据权利要求5所述的生物样品制备方法，其特征在于：所述步骤S2中，冷冻替代程序为：将温度从-90℃逐渐升温到0℃。
7.根据权利要求1、2、4、6中任意一项所述的生物样品制备方法，其特征在于：所述S4步骤的具体步骤如下：
S41:将装有样品的容器转移到0摄氏度的载台上，并利用预冷的含有戊二醛的丙酮溶液漂洗两次；
S42:用梯度丙酮溶液对漂洗好的样品进行复水处理，依次在每个梯度溶液中浸泡；
S43:将样品分别在固定液I和固定液II中浸泡；
S44:将样品在HEPES缓冲液浸泡；
S45:将样品在PBS缓冲液浸泡。
8.根据权利要求1、2、4、6中任意一项所述的生物样品制备方法，其特征在于：所述步骤S5的具体步骤如下：
S51:将样品转移到锇酸固定液A中，并置于0摄氏度的载台上，然后用双蒸水漂洗；
S52:将样品转移至硫代对称二氨基脲中孵育，然后用双蒸水漂洗；
S53:将样品转移至锇酸中，然后漂洗。
9.根据权利要求8所述的生物样品制备方法，其特征在于：所述步骤S6的具体步骤如下：
S61：将样品转移至醋酸双氧铀中染色，然后漂洗。
10.根据权利要求1或9所述的生物样品制备方法，其特征在于：所述步骤S7的具体步骤如下：
S71：将样品转移至铅染液中孵育，然后漂洗。
11.根据权利要求10所述的生物样品制备方法，其特征在于：所述步骤S71中，铅染液的配制：将天冬氨酸溶解于水中配制成天冬氨酸缓冲液，将硝酸铅溶解于天冬氨酸缓冲液中，调节pH值到5.5。
12.根据权利要求7所述的生物样品制备方法，其特征在于：所述步骤S41中，所述梯度丙酮溶液为用水和纯丙酮配制的浓度在95％～70％之间的若干个浓度的溶液，且每个浓度的溶液中均含有戊二醛。
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