CN108139408A - 蛋白质保持扩展显微法 - Google Patents
蛋白质保持扩展显微法 Download PDFInfo
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- CN108139408A CN108139408A CN201680051406.XA CN201680051406A CN108139408A CN 108139408 A CN108139408 A CN 108139408A CN 201680051406 A CN201680051406 A CN 201680051406A CN 108139408 A CN108139408 A CN 108139408A
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Abstract
本发明提供被称为蛋白质保持ExM(proExM)的方法,其中使用交联分子将蛋白质,而不是标记物锚定到可溶胀的凝胶。这种proExM策略可用于进行免疫染色细胞和组织以及表达FP的样品的纳米级成像,因为即使当经受非特异性蛋白质水解消化时,来自直接锚定到凝胶的基因编码荧光蛋白和/或常规荧光标记二级抗体和链霉亲和素的荧光信号得以保持。
Description
相关申请
本申请要求2015年8月7日提交的美国临时申请序列号62/202,423的优先权益,其内容以引用的方式整体并入本文。
政府资助
本发明是在由Hertz Foundation、NYSCF、NSF和Rehabilitation Institute ofChicago授予的NYSCF-R-NI10和由NIH和Cargill Fund Bioengineering Fund授予的1-U01-MH106011的政府支持下完成的。政府对本发明拥有某些权利。
背景技术
扩展显微法(ExM)可以~70nm横向分辨率来对厚的保存标本成像。使用ExM,通过在成像之前物理扩展生物样本来规避光学衍射极限,从而使亚衍射限制结构达到可通过常规衍射限制显微镜来观察的尺寸范围。ExM可以以衍射限制显微镜的体素率但是以超分辨率显微镜的体素尺寸来对生物标本成像。扩展样品是透明的,并且与水的折射率匹配,因为扩展材料是>99%的水。原始ExM协议通过使用可凝胶锚定的荧光团标记感兴趣的生物分子来运作。然后,在样品中合成可溶胀的聚电解质凝胶,从而它将标记物并入。最后,用非特异性蛋白酶处理样品以使其机械特性均匀,接着在水中透析以介导聚合物-样本复合物的均匀物理扩展。除了可凝胶锚定的标记物以外,ExM所需的所有化学品都可以购买,这种标记物需要定制合成并且对研究人员采用所述方法造成了障碍。ExM协议的另一个缺点是基因编码的荧光团不能在没有抗体标记的情况下成像。另外,ExM不能在凝胶中保持天然蛋白质,并且使用不能广泛获得的定制试剂。因此,需要利用ExM来设计将样品内的核酸和蛋白质保持在原位并且加以成像的新方法。
发明概述
本发明提供被称为蛋白质保持ExM(proExM)的方法,其中使用交联分子将蛋白质,而不是标记物锚定到可溶胀的凝胶。这种proExM策略可用于进行免疫染色细胞和组织以及表达FP的样品的纳米级成像,因为即使当经受非特异性蛋白质水解消化时,来自直接锚定到凝胶的基因编码荧光蛋白和/或常规荧光标记二级抗体和链霉亲和素的荧光信号得以保持。
在一个实施方案中,本发明提供用于感兴趣的样品的蛋白质的保持和成像的方法,其包括以下步骤:使样品内的蛋白质与双官能交联剂缀合;使样品包埋在可溶胀材料中,其中样品内的蛋白质锚定到可溶胀材料;使样品经受消化;使可溶胀材料溶胀以形成扩展样品;并且使感兴趣的样品成像。
附图简述
从如在附图中示出的本发明的优选实施方案的以下更加具体的描述中将明白本发明的前述和其它目的、特征以及优点,在附图中相似元件符号指代不同视图中的相同部分。附图未必按比例绘制,而是将重点放在示出本发明的原理上。
专利或申请文件含有至少一幅彩色附图。在提出请求并支付必要费用后,本事务所将提供具有彩色附图的本专利或专利申请公布的副本。
图1:38g/100mL丙烯酸钠储备溶液:合适(澄清,左)和低纯度(淡黄色,右)。
图2:是凝胶腔室的示意图。
图3a-图3c:使用高压灭菌版的组织破坏方案,扩展之前(a)和之后(b),Thy1-YFP-表达大脑组织的落射荧光图像(仅绿色通道)。扩展后共焦图像(c)。扩展组织是使用针对绿色荧光蛋白(GFP,绿色)、GAD65/67(红色)和SV2(蓝色)的一级抗体染色的抗体。比例尺:(a)50um,(b)500um扩展前(2.2mm扩展后),(c)10um扩展前(44um扩展后)。
图4a到图4l。荧光蛋白(FP)和抗体荧光信号在FP融合蛋白的proExM和proExM中的保持。(a)活HEK293FT细胞(上排)和proExM处理后的相同细胞(下排)中所选FP-组蛋白融合蛋白的代表性图像;iRFP被表达为具有核定位序列(NLS)的N端融合物。(b)在proExM处理后,如图a中的实验的量化荧光(斜线阴影条;平均值±标准偏差;各自n=4次转染重复)。空心条,这些荧光团的亮度的文献值,相对于EGFP的亮度来归一化。(c)在proExM处理后,对于小鼠脑切片中与抗体缀合的所选染料的荧光保持(平均值±标准偏差,各自n=3个样品)。(d)锚定步骤后免疫染色的微管的超分辨率结构照明显微术(SR-SIM)图像相比于(e)用旋转盘共焦显微镜获得的相同样品的扩展后图像。(f)proExM相比于SIM图像的作为测量长度的函数的均方根(RMS)长度测量误差(蓝线,平均值;阴影面积,标准偏差;n=4个样本)。(g)mClover-α-微管蛋白融合的共焦图像。HeLa细胞在本图的其余部分使用。图(i和ii)是(g)中加框区域的放大视图。线切在图h、i中量化。(h,i)中的红色实线表示用于确定半极大处全宽度的高斯拟合(FWHM;用红色箭头表示)。(j)mEmerald-网格蛋白融合的共焦图像(左)和加框区域中单个CCP的放大视图(右)。(k)网格蛋白(融合到mEmerald,绿色)和角蛋白(mRuby2,红色)的双色proExM。(l)肌动蛋白(mRuby2,红色)和桩蛋白(mEmerald,绿色)融合的双色proExM图像。图(i和ii)是(f)中加框区域的放大视图。比例尺:(a)5μm,(d)5μm,(e)5μm(扩展后物理尺寸,20.5μm),(g)5μm(21.5μm),(i-ii)1μm;(j)10μm(42.6μm),插图200nm;(k)1μm(4.3μm),(l)5μm(21.5μm),(i-ii)1μm。
图5a至图5p。不同哺乳动物组织类型中proExM的验证。(a-d)Thy1-YFP小鼠脑(a)和波形蛋白免疫染色的小鼠胰腺(b)、脾(c)以及肺(d)的扩展前(上)和扩展后(下)样品的低放大倍数的广角图像。(e)在用超分辨率结构化照明显微术(SR-SIM)(绿色)和使用具有重叠失真矢量场(白色箭头)的常规共焦显微术的proExM(紫色)成像的Thy1-YFP小鼠脑中Tom20的复合荧光图像。(f)示出(a)中的加框区域的扩展前SR-SIM图像。(g)(f)的扩展后共焦图像。(h)Thy1-YFP小鼠脑中Tom20染色的proExM相比于扩展前SR-SIM的作为测量长度的函数的RMS长度测量误差(蓝线,平均值;阴影面积,标准偏差;n=3个小鼠大脑皮层样品)。(i)用失真矢量场重叠扩展之前(绿色)和之后(紫色)小鼠胰腺中波形蛋白的高放大倍数广角荧光合成图像(白色箭头,参见方法)。(j)显示(i)中加框区域的扩展前的广角图像。(k)(j)的扩展后图像。(l)针对(b-d)中不同组织类型,proExM相比于广角扩展前图像,作为测量长度的函数的均方根(RMS)长度测量误差(蓝线,平均值;阴影面积,标准偏差;n=来自胰腺、脾和肺的3个样品)。(m)在用超分辨率结构化照明显微术(SR-SIM)(绿色)和使用具有重叠失真矢量场(白色箭头)的常规共焦显微术的proExM(紫色)成像的小鼠胰腺中波形蛋白的复合荧光图像。(n)示出(m)中的加框区域的扩展前SR-SIM图像。(o)(n)的扩展后共焦图像。(p)胰腺中波形蛋白染色的proExM相比于扩展前SR-SIM的作为测量长度的函数的RMS长度测量误差(蓝线,平均值;阴影面积,标准偏差;n=来自2个样品的4个视野)。比例尺:(a)顶部200μm,底部200μm(扩展后物理尺寸,800μm),(b-d)顶部500μm,底部500μm(分别2.21mm、2.06mm、2.04mm),(e,f)10μm,(g)10μm(40μm),(i)10μm,(j)5μm,(k)5μm(20.4μm),(m)5μm,(n)5μm,(o)5μm(20.65μm)。
图6a到图6h。哺乳动物脑回路的proExM。(a)在病毒注射恒河猴皮层中的GFP荧光的广角图像。(b)(a)的扩展后广角荧光图像。(c)(b)分区的共焦显微镜图像的体绘制。插图显示示出树突棘的放大的(c)中加框区域。(d)低放大的广角荧光成像显示表达病毒递送的Brainbow3.0的免疫染色的小鼠海马体。(e)来自(e)的样品的扩展后广角图像。(f)来自(e)中加框区域的膜标记的Brainbow3.0神经元的扩展之后的MIP高分辨率共焦显微镜图像。(g)扩展前共焦图像,显示(f)中加框区域的一个光学部分。(h)(g)的扩展后图像。比例尺:(a)100μm,(b)100μm(扩展后物理尺寸,413μm);(c)300μm x 254μm x 25μm,(c)(i)1μm,(d)500μm,(e)500μm(1980μm);(f)5μm,(g)5μm(19.8μm);(h)50μm(198μm)。
图7.具有蛋白质保持的扩展显微术的工作流程。本文探讨了三种基本的样品处理工作流程。顶部,在室温下,在AcX处理和随后ExM处理(凝胶化、蛋白酶K处理和水扩展)之前,使用常规免疫染色方案,将样品化学固定并且使用抗体染色。中间,表达荧光蛋白(FP)的样品在AcX处理和随后的ExM处理之前化学固定(并且任选透化)。底部,用AcX处理样品,然后凝胶化,并且然后用温和的均化程序(例如,碱性水解和变性,或使用LysC消化)处理,并且最后在扩展状态下进行抗体染色。
图8a到图8h。扩展后抗体递送,在表位保存均化后。(a,b)扩展前(a)和高压灭菌处理和抗体染色后(b),Thy1-YFP表达小鼠脑半球切片的广角荧光图像。(c-h)使用不同破坏方法和抗体处理的Thy1-YFP表达小鼠脑的皮层的共焦显微照片,使用抗-GFP(绿色,染色YFP)作为参考。(c)高压灭菌法,然后针对bassoon(蓝色)和homer(红色)染色。(d)高压灭菌,然后进行髓磷脂碱性蛋白染色。(e)高压灭菌,然后波形蛋白(红色)和胶质纤维酸性蛋白(蓝色)染色。(f)在高压灭菌(i)或LysC(ii),或者在LysC均化后应用二级抗体(其中一级抗体使用AcX先前锚定到凝胶)处理后的Lamin A/C染色(g-h)使用高压灭菌法,针对Tom20(i)和YFP(ii,在底部图中红色通道中显示,因为内源性YFP是绿色)的凝胶化(g)前相比于破坏后(h),和在使用LysC破坏后针对homer(红色)和PSD-95(蓝色)(iii)的染色的比较。比例尺:(a)1mm,(b)1mm(3.96mm),(c-h)5μm(~21μm)。
图9a到图9e。使用AcX和LysC温和消化法处理的Thy1-YFP小鼠脑切片的扩展前和扩展后图像。(a)扩展前广角图像。(b)扩展后广角图像。箭头指示图像(c-e)的定位。围绕切片的明亮边缘是凝胶-空气界面处散射的结果。(c)海马体中感兴趣的选择区域的扩展前共焦图像。(d)与(c)中相同的选择区域的扩展后共焦图像。(e)如(d)中所示相同的选择区域的扩展后DIC图像。比例尺:(a)1mm,(b)4mm(扩展后单元),(c)5μm,(d-e)20μm(扩展后单元)。
图10和图10b。使用高压灭菌和LysC方法的不完全均化。Thy1-YFP表达小鼠大脑皮层的荧光图像,其中在高压灭菌处理和抗体染色后使用共焦成像使用抗-GFP染色的YFP,显示不存在于成像体积表面的不连续轴突(a),以及在LysC处理和抗体染色后使用广角成像,显示包含白质纤维束的扩展区域中的缺陷(b)。比例尺;(a)5μm(~20μm),(b)0.5mm(~2mm)。
图11a到图11f。使用高压灭菌、LysC和凝胶化前抗体处理的免疫染色法的比较。Thy1-YFP表达小鼠大脑皮层的共焦图像,凝胶化前免疫染色,然后AcX处理、凝胶化和蛋白酶K消化(proExM),列(i)。AcX处理和凝胶化,然后高压灭菌处理,列(ii),或LysC消化列(iii)后经免疫染色的Thy1-YFP脑样品。高压灭菌和LysC样本均具有抗-GFP(绿色)染色的YFP,除TOM20((a)排)、homer(红色)和bassoon(蓝色)((b)排)、homer(红色)和突触后密度95(PSD95,蓝色)((c)排)、谷氨酸脱羧酶(GAD)65/67((d)排)、髓磷脂碱性蛋白(MBP,(e)排)以及波形蛋白(红色)和胶质纤维酸性蛋白(GFAP,蓝色)((f)排)之外。比例尺;5μm(~20μm)。
图12a到图12g。proExM后HEK293FT细胞中EGFP和EYFP荧光的保持的对照实验。(a)在活HEK293FT细胞中以及在不具有(顶部)或具有(底部)AcX处理的情况下proExM处理之后的EGFP-H2B融合的代表性图像。比例尺20μm。(b)相对于活细胞,在不具有(左)或具有(右)AcX处理的情况下在proExM处理后保持的EGFP荧光的百分比(平均值±标准偏差,n=4)。(c)在活HEK293FT细胞中(左上)以及在收缩(左上)和完全扩展凝胶(下)中的proExM处理之后的EGFP-H2B融合的代表性图像。比例尺5μm。(d)相对于活细胞,在收缩(左)和完全(右)扩展凝胶中proExM处理之后保持的EGFP荧光的百分比(平均值±标准偏差,n=4个样品)。(e)在活(虚线,n=8个细胞)和proExM处理的完全扩展HEK293FT细胞(实线,n=7个细胞)中测量的在广角照明(475/34nm,~60mW/mm2光功率)下的EGFP的光漂白的归一化曲线。(f)在活(虚线,n=14个细胞)和proExM处理的完全扩展HEK293FT细胞(实线,n=5个细胞)中测量的在广角照明(512/10nm,~8.4mW/mm2光功率)下的EYFP的光漂白的归一化曲线。(g)在4℃下在1x PBS中长期储存后,proExM处理的HEK293FT细胞中EGFP和EYFP荧光的保持(n=3个样品)。
图13a到图13c。S-亚硝基化的ProExM成像。(a)一级神经元培养物中使用抗-微管蛋白染色的微管蛋白纤维的ProExM。(b)结合到通过SNOTRAP方法化学标签的生物素化的半胱氨酸S-亚硝基化蛋白质的荧光标记链霉亲和素的ProExM。(c)(a)和(b)的彩色复合图像(微管蛋白,红色;SNOTRAP,绿色)。
图14a到图14g。在proExM中选择的光可切换和光可活化的FP的性能。(a)活HEK293FT细胞(活,每个FP的上部图像)和proExM处理后相同的细胞(proExM,每个FP的下部图像)中选择的光可切换/光可活化的FP-组蛋白融合的代表性图像。(b)proExM处理之前(活,空心条)和之后(proExM,交叉阴影条,平均值±标准偏差,各自n=4个转染复制物)的HEK293FT细胞中所选择的FP-组蛋白融合的荧光。相对EGFP,所选择的FP的荧光归一化至它们分子亮度。(c)proExM后,HEK293FT细胞的核中未转化的H3.3-Dendra2的100个连续帧的平均强度图像,由488nm激光器激发。(d)衍生自c中细胞的10,000连续帧的PALM图像,其使用低功率连续405nm激光器激发来进行光转换。根据它们半极大处全宽度定位,使用高斯掩模估计显示196,441个检测到的颗粒。针对H3.3-Dendra2融合,平均和中位定位误差为23.3nm。(e)来自mEos2-α-微管蛋白的光子总数的分布(平均值196.6,中位数169.6)。(f)针对mEos2-α-微管蛋白融合,平均和中位定位误差分别为26.1nm和25.9nm。(g)衍生自表达mEos2-α-微管蛋白的proExM处理的HeLa细胞的15,000连续帧的PALM图像,其使用低功率连续405nm激光激发来进行光转换。根据它们半极大处全宽度定位,使用高斯掩模估计显示315万检测到的颗粒。比例尺:(a)10μm,(c-d,g)2.2μm(扩展后物理尺寸,10μm)。
图15a到图15e。Thy1-YFP小鼠脑切片和具有Brainbow3.0荧光蛋白并且使用proExM处理的小鼠脑扩展前和扩展后图像。(a)Thy1-YFP脑切片的扩展前广角图像。(b)来自a的切片的扩展后广角图像。(c)Brainbow3.0小鼠脑组织中膜结合GFP的扩展后最大强度投影图像(Z中~10μm)。(d)来自c的图像的一个Z切片。(e)Brainbow3.0小鼠组织中膜结合GFP和膜结合mCherry的双色成像的扩展后成像。比例尺:(a),(b)500μm(20.5μm)。(c-e)5μm(~20μm)。
图16a到图16i。优化固定脑组织中AcX的穿透深度。(a)用于从组织切片一侧来测量NHS-酯混合物(99%AcX+1%NHS-生物素,其具有与AcX相似的分子量和电荷)的穿透深度的腔室测定。在使用NHS-酯混合物处理过夜后,取回切片,洗涤并且使用荧光团缀合的链霉亲和素处理以使NHS-酯混合物的穿透可视化。(b)在腔室测定条件下染色的100-μm厚的小鼠脑切片的代表性图像。比例尺1mm。(c)沿着在b中表示为白色虚线的线切割的荧光强度。强度从最大值下降到其值的一半的距离(D1/2)是NHS-酯穿透深度的特征长度。(d,e)在测试的所有pH水平下,使用基于MES的盐水(MBS;100mM MES+150mM NaCl)染色产生比基于磷酸盐的盐水(PBS)显著改善的NHS-酯穿透深度。比例尺1mm。(f,g)在4℃下染色产生比室温下适当更大的穿透深度。比例尺1mm。(h)在proExM之前(左)和之后(右),在500-μm厚的Thy1-YFP小鼠脑切片中天然YFP荧光的代表性图像。比例尺1mm(扩展前单元)。(i)共焦成像展示在使用MBS,pH6.0过夜AcX处理后,500-μm厚切片的中心处的YFP荧光保持。比例尺100μm(扩展后单元)。
发明详述
除非上下文不合适,否则本文所用术语“一(a)”、“一(an)”和“所述(the)”意指“一或多个”且包括复数个。
通过引用并入本文并且作为附录A附属的国际专利申请序列号PCT/US15/16788教导了常规显微镜的分辨率可通过物理扩展样本(称为“扩展显微”(ExM)的方法)来增加。在ExM中,化学固定并且透化的组织灌注有可溶胀材料,经受聚合并且使用蛋白酶处理组织-聚合物复合物以使其机械特性匀化。接着,水中透析产生各向同性~4倍的线性扩展,从而实现超分辨率的具有衍射限制的显微镜,从而实现快速图像采集和大视野(Chen等人,Science,347,543(2015))。ExM的优点包括组织透明化,分辨率提高以及由于z轴上的样本扩展导致的切片误差更高的容差。
本发明提供被称为蛋白质保持ExM(proExM)的ExM的变体,其中使用交联分子将蛋白质,而不是标记物锚定到可溶胀的凝胶。即使当经受来自初始ExM方案的非特异性蛋白质水解消化时,来自直接锚定到凝胶的基因编码荧光蛋白和传统荧光标记二级抗体和链霉亲和素的荧光信号得以保持。proExM是用于制备用于成像的样品的标准组织学方法的延伸。
这种蛋白质保持ExM(proExM)策略可用于进行免疫染色的细胞和组织(图7,顶部)以及表达各种FP的样品(图7,中部)的纳米级成像。ProExM变体可支持扩展后抗体递送,从而潜在地增加染色的亮度和抗体进入(图7,底部)。
在一个实施方案中,本发明提供感兴趣的生物样品的蛋白质的保持和成像的方法,其包括以下步骤:
(a)使样品内的蛋白质与双官能交联剂缀合;
(b)使样品包埋在可溶胀材料中,其中样品内的蛋白质锚定到可溶胀材料;
(c)使样品经受消化;
(d)溶胀可溶胀材料以形成扩展样品;以及
(e)使感兴趣的样品成像。
在一个实施方案中,双官能交联剂包含对样品内蛋白质上官能团(例如,伯胺或硫氢化物)有反应性的基团。用这种双官能交联剂允许样品内蛋白质直接锚定到或并入到可溶胀材料中。在一个实施方案中,双官能交联剂是杂-双官能交联剂。杂-双官能交联剂在间隔物臂(即,分离反应性基团的原子、间隔物或接头)的任一端部具有不同的反应性基团。这些试剂不仅允许具有相应的靶标官居能团的分子的单个步骤缀合,它们还允许将不期望的聚合或自缀合最小化的顺序(两个步骤)缀合。
在一个实施方案中,双官能交联剂包含蛋白质反应性化学部分和凝胶反应性化学部分(即,可溶胀材料反应性化学部分)。蛋白质反应性化学基团包括但不限于例如,可以与蛋白质或肽上的氨基或羧酸基团反应的N-羟基琥珀酰亚胺(NHS)酯、硫醇、胺、马来酰亚胺、亚氨酸酯、吡啶基二硫醇,酰肼、邻苯二甲酰亚胺、双吖丙啶、芳基叠氮化物、异氰酸酯或羧酸,在一个实施方案中,蛋白质反应性基团包括但不限于N-琥珀酰亚胺酯、五氟苯基酯、羧酸或硫醇。凝胶反应性基团包括但不限于乙烯基或乙烯基单体,诸如苯乙烯及其衍生物(例如二乙烯基苯)、丙烯酰胺及其衍生物、丁二烯、丙烯腈、乙酸乙烯酯或丙烯酸酯和丙烯酸衍生物。
在一个实施方案中,将蛋白质直接锚定到任何可溶胀材料的化学品是6-((丙烯酰基)氨基)己酸的琥珀酰亚胺基酯(丙烯酰基-X,SE;缩写“AcX”;Life Technologies)。用AcX处理修饰具有丙烯酰胺官能团的蛋白质上的胺。丙烯酰胺官能团允许蛋白质在原位合成时锚定到可溶胀材料。
在一个实施方案中,感兴趣的样品的蛋白质可使用点击化学在单独的步骤中用蛋白质反应性基团和凝胶反应性基团修饰。点击化学也称为标签,是一类生物相容性反应,主要用于连接选择的底物和特定生物分子。在此方法中,感兴趣样品的蛋白质用包含点击基团的蛋白质反应性基团处理,并且然后用包含互补性点击基团的凝胶反应性基团处理。互补性基团包括但不限于叠氮基团和终端炔烃(参见例如,H.C.Kolb;M.G.Finn;K.B.Sharpless(2001)."Click Chemistry:Diverse Chemical Function from a FewGood Reactions".Angewandte Chemie International Edition.40(11):2004–2021,其通过引用并入本文)。
在一些实施方案中,锚定到如本文所述的在整个样品内灌注的可溶胀材料的天然蛋白质可保持表位功能性,并且如果ExM的非特异性蛋白水解被修饰的凝胶后均化处理替换,则可以在扩展后进行标记。这些方法可以克服在天然组织拥挤的环境中递送抗体所固有的限制15–19。例如,紧密排列的表位可能与致密组织中的抗体结合不佳,但扩增后更易进入抗体(图8)。
在一个实施方案中,消化包括在富含碱性洗涤剂的缓冲液中在高压灭菌中处理凝胶包埋的组织(例如Thy1-YFP小鼠脑样品)1小时(图8a,显示内源性YFP预处理;图8b,显示使用抗GFP的扩展后标记)。在另一个实施方案中,消化包括将凝胶包埋的组织暴露于LysC,其在Lys残基处切割蛋白质(与非特异性蛋白酶K相反)4,5(图9)。发现抗体确实可以在扩展后成功递送(图8c-e)。
在另一个实施方案中,本发明提供了将直接蛋白质锚定的便利性与强酶(例如蛋白酶K)消化相结合的方法。用AcX处理,然后用标准的ExM工作流程(包括蛋白酶K消化),可高效保存扩展凝胶中的荧光(65%±5%保存;平均值±标准偏差;n=4;图4a、5b和图12)。
系统地检查了proExM工作流程中各种荧光蛋白(“FP”)的荧光持续性。选择了20种从蓝色到近红外光谱范围的广泛使用的FP(表1)。
表1.proExM中选择的FP的性能。
a来自Addgene质粒37132的mKate2基因与Addgene质粒37133中的LSSmOrange基因交换。
b克隆为具有核定位序列的N末端融合。
c由于EYFP对用于收缩凝胶的高Cl-特别敏感48,所以在完全扩展的凝胶中测量EYFP荧光的保持。
选择的FP与组蛋白蛋白融合并且在人胚胎肾(HEK293FT)细胞中表达。比较相同细胞的活培养物的图像相比于proExM后图像(图4a)。proExM(各自n=4个样品;图4a、4b,表1)后,大多数FP保持其活荧光强度的50%以上,与初始ExM方案中小分子荧光团的持续性相当1。
已经看到,即使在强消化过程之后,FP仍然持续充分报告信号,确定proExM锚定并且保持荧光缀合的二级抗体的荧光。凝胶化和消化后,带有多种小分子荧光团的二级抗体标记的样本保持其开始亮度的~50%(各自n=3个样品;图4c;表2)。
表2.在proExM中选择的二级抗体染料的性能。
因此,proExM允许使用商业二级抗体,而不要求定制制剂的需要。
除了抗体之外,还观察到来自荧光标记的链霉亲和素的信号的保存。使用链霉亲和素,被设计来使用先前开发的化学方法SNOTRAP8来捕获半胱氨酸-S-亚硝基化的探针得以成像,从而证明用proExM的S-亚硝基化信号的成像(图13)。此方案也指出了将其他蛋白酶抗性标签锚定到聚合物上,随后凝胶化、消化、扩展以及成像作为潜在的广义策略的可能性。
尽管消化步骤保存了扩展样本的纳米级各向同性,但是通过在proExM之前的超分辨率结构化照明显微镜(SR-SIM)(图4d)和proExM之后的共焦成像(图4e),对培养细胞中的免疫染色微管进行成像来验证proExM。量化介于0微米到20微米之间长度范围内proExM后特征测量的均方根(RMS)误差,并且发现RMS误差为测量距离的~1%-2%(图4f)。
进行proExM,然后共焦显微术,以对培养的HeLa细胞中带有基因编码的荧光团(即,未染色)的几种融合蛋白进行成像。检查了微管蛋白、网格蛋白和角蛋白的融合(图4g-k),其通常用作定型结构以展示细胞的超分辨率成像9–12。微管蛋白-mClover融合在67±8nm的半极大处全宽度(FWHM)处呈现微管(3个样品中n=16个微管)(图4h,i),表明优于70nm的分辨率11。在HeLa细胞中的网格蛋白-mEmerald也被成像,从而获得了在凹坑中间的空位的极好定义(图4j)。包含筛选中的基因编码荧光团中的两个,即mEmerald和mRuby2的融合蛋白质的双色proExM成像获得如预期的极好的图像质量(角蛋白-mRuby2和网格蛋白-mEmerald,图4k;桩蛋白-mEmerald和肌动蛋白-mRuby2,图4l)。在proExM期间检查了四个光可切换FP的稳定性(图14,表3)。
表3.在proExM中选择的光可切换和光可活化的FP的性能。
表达组蛋白2B-Dendra和mEos2-微管蛋白融合的细胞使用PALM显微镜成像(图14),证明与PALM相容的光切换荧光团的保存。
为了评估各种三维组织中proExM的性能,对四种不同的小鼠组织类型(脑、胰腺、肺以及脾,图5a-d)进行proExM。在神经元的稀释子集中,Thy1启动子之下表达YFP(Thy1-YFP)的小鼠脑在如培养细胞所述的用蛋白酶K处理后在没有毫米级尺度下的失真的情况下扩展(图5a,顶部相比于底部)。胰腺、脾和肺具有不同于脑的机械特性(例如更多的结缔组织),这阻碍了室温蛋白酶K消化后的扩展。中间纤维波形蛋白被抗体染色作为结缔组织的标志物以检查这些不同组织类型中的扩展的各向同性。proExM允许胰腺、肺和脾组织扩展,在毫米长度尺度内具有良好的组织形态保存(图5b-d,顶部相比于底部)。proExM之前(图5e,5f)相比于之后(图5e,5g)的组织的高分辨率衍射限制显微术显示了proExM的分辨率提高。扩展的各向同性通过测量胰腺、肺和脾组织中微尺度(<100μm)的proExM后特征测量的均方根(RMS)误差来量化。在这个长度尺度内,RMS误差很小(1%-3%的测量距离),并且在所有三种组织类型中相似(图5h)。
为了检查纳米级扩展的各向同性,对胰腺中的波形蛋白染色进行SR-SIM(图5i,5j)和proExM共焦成像(图5i,5k)。同样,观察到在0微米与25微米之间的测量的测量长度的大约1%-5%的小RMS误差(图5l,从2个样品,n=4个视野)。对用线粒体标记物Tom20抗体染色的小鼠大脑皮质组织进行类似分析,并且在室温下用蛋白酶K消化的proExM处理之前用SR-SIM成像(图5m,5n)并且之后共焦成像(5o)。这种组织类型的RMS误差在测量长度的1%-3%之间,在0微米与40微米之间(图5p,n=3个样本)。
在生物学中常规使用表达FP的转基因动物以及在病毒基因递送后表达FP的动物,用于标记完整组织和生物体中的蛋白质和细胞。proExM用于在完整的哺乳动物脑组织(包括小鼠(图15)和恒河猴(图6a-c)的大脑)中表达的FP的可视化,从而获得在扩展后显示最小宏观尺度失真的图像(例如,比较图6a对6b)。在常规共焦显微镜上使用高倍镜头,扩展后树突棘形态很容易分辨,甚至可以看到细的脊椎颈(图6c插图,箭头)。
proExM用于对表达病毒式递送的Brainbow3.013,14的小鼠脑回路进行成像,其用膜锚定的FP的随机组合来标记神经元。这些FP在抗原性方面不同以允许随后通过抗体扩增。在proExM后,抗体染色和形态学被保存在脑组织中(图6d对6e)。共焦成像允许Brainbow样品的大体积、多色超分辨成像(图6f)。扩展之前(图6g)和之后((图6h)共焦图像的并行比较显示扩展前轴突和树突太接近以致于难以分辨如何能够在扩展后清晰地分辨(图6g、h)。
使用标准方法递送的荧光蛋白和荧光抗体也保持在凝胶中,并且在非特异性蛋白水解处理之后还显示荧光信号。proExM的多色彩、大容量能力通过扩展Brainbow样品来证明,这对于回路映射可能是有用的。内源性荧光的保存允许使用转基因动物、病毒表达载体和FP的转染,所有这些都没有免疫染色。
使用proExM处理的样品是光学透明的,并且与水的折射率匹配1。这允许在传统的荧光显微镜上进行深入到样品中的超分辨率成像,仅受物镜工作距离的限制。
在一个实施方案中,本发明提供了将蛋白质锚定到扩展显微术(ExM)的可溶胀凝胶中,随后进行更温和的破坏处理,其使对单个蛋白质的损伤最小化,从而允许在扩展之后在蛋白质上进行染色和其他处理。
与先前描述的ExM方法(其中所有染色必须在扩展之前在天然完整组织状态下进行)相比,本发明允许染色在扩展状态下进行,其中天然蛋白转移到扩展凝胶的准体外环境中。不希望受限于任何特定理论,据信这种简化的化学环境减轻了限制生物染色方法的许多问题,包括空间位阻和扩散通路以及潜在地还有自发荧光和非特异性结合。因此,提供了以比当前染色方法所需的更少的优化来进行厚组织标本的快速染色、更高的染色强度和潜在更好的挑战性靶标染色。本发明还能够使用与天然组织环境不相容的探针,以及其他潜在的应用。
在一个实施方案中,本发明提供用于将蛋白质直接锚定至任何可溶胀材料的化学品,如国际专利申请序列号PCT/US15/16788中所述。在一个实施方案中,将蛋白质直接锚定到任何可溶胀材料上的化学品是6-((丙烯酰基)氨基)己酸的琥珀酰亚胺酯,其带有与蛋白质上的赖氨酸残基反应的琥珀酰亚胺酯部分和合成时反应到可溶胀材料中的丙烯酰基部分。
在另一个实施方案中,本发明提供了用于组织破坏的方法,该方法被设计成允许组织-凝胶复合物的均匀扩展,同时在分子水平上最小程度地破坏组织,实质上使组织破碎并扩展组织,而不是强烈溶解它。在一个实施方案中,本发明提供了单独或组合的以下各项的应用:不含酶的洗涤剂和高温,裂解除蛋白质以外的生物分子的酶,以比蛋白酶K更高的特异性或更小程度裂解蛋白质的酶,用于脂质提取的非水性溶剂,蛋白质和其他生物分子(包括核苷酸、多糖和脂质)的受控化学裂解。这也包括强酶消化,只要所研究的蛋白质对这种处理是稳健的。
在一个实施方案中,本发明提供了用于扩展状态下的组织的染色和其他生物化学表征的方法。
如本文所用,术语“感兴趣的样品”通常是指但不限于生物、化学或生物化学样品。在一个实施方案中,感兴趣的样品是生物样品。生物样品包括但不限于:组织、细胞或其任何组分,肿瘤或任何器官的全部或一部分,包括但不限于脑、心脏、肺、肝、肾、胃、结肠、骨骼、肌肉、皮肤、腺体、淋巴结、生殖器、乳房、胰腺、前列腺、膀胱、甲状腺以及眼睛。
在一个实施方案中,感兴趣的样品可优选用可检测标记进行标记或标志。典型地,标记物或标签将化学地(例如,共价地、氢键合或离子键合)结合至样品或其组分,例如一种或多种蛋白质。可检测标记可针对特定靶标(例如,生物标志物或分子类别)具有选择性,如可以用抗体或其他靶标特异性结合剂完成的。可检测标记优选包含可见组分,如典型的染料或荧光分子;然而,标记所使用的任何信号传导手段也是可以预期的。使感兴趣的样本与可检测标记接触产生“经标记的感兴趣的样品”。
例如,荧光标记的感兴趣样品是通过诸如但不限于免疫荧光、免疫组织化学或免疫细胞化学染色的技术来标记以帮助显微镜分析的感兴趣样品。因此,可检测标记优选化学连接到感兴趣的样品或其靶标组分。在一个实施方案中,可检测标记是抗体和/或荧光染料。抗体和/或荧光染料还包含将样品连接或交联到可溶胀材料(诸如水凝胶)的物理、生物或化学锚定剂或部分。
标记的样品还可包含多于一个的标记。例如,每个标记可具有特定或可区分的荧光特性,例如可区分的激发和发射波长。此外,每个标记可以具有不同的靶标特异性结合剂,其对样品中的特定和可区分的靶标或组分具有选择性。
如本文所用,术语“凝胶”或“可溶胀材料”可互换使用,以通常指当与液体(诸如水或其他溶剂)接触时扩展的材料。在一个实施方案中,可溶胀材料在三维上均匀扩展。另外地或可选地,材料是透明的,使得在扩展时光可穿过样品。在一个实施方案中,可溶胀材料是可溶胀的聚合物或水凝胶。在一个实施方案中,可溶胀材料由其前体原位形成。例如,可以使用一种或多种可聚合材料、单体或低聚物,诸如选自由包含可聚合烯键式不饱和基团的水溶性基团组成的组的单体。单体或低聚物可包含一种或多种取代或未取代的甲基丙烯酸酯、丙烯酸酯、丙烯酰胺、甲基丙烯酰胺、乙烯醇、乙烯胺、烯丙基胺、烯丙醇,包括其二乙烯基交联剂(例如N,N-亚烷基双丙烯酰胺)。前体还可以包含聚合引发剂、加速剂、抑制剂、缓冲剂、盐以及交联剂。
在一个实施方案中,可溶胀聚合物是聚丙烯酸酯及其共聚物或交联共聚物。可选地或另外地,可通过化学交联水溶性低聚物或聚合物可原位形成可溶胀材料。因此,本发明设想将可溶胀材料的前体(诸如水溶性前体)加入到样品中并且使前体原位可溶胀。
如本文所用,将样品在可溶胀材料中“凝胶化”或“包埋”可互换使用,指的是样品用可溶胀材料的渗透(诸如灌注、输注、浸泡、加入或其他混合),优选通过加入其前体。可选地或另外地,将样品包埋在可溶胀材料中包括在整个样品中渗透一种或多种单体或其他前体,并使单体或前体聚合和/或交联以原位形成可溶胀材料或聚合物。以这种方式,感兴趣的样品被包埋在可溶胀材料中。
在一个实施方案中,用包含水可溶胀材料的水溶性前体的组合物渗透感兴趣的样品或标记的样品,并且使前体反应以原位形成水可溶胀材料。
在某些实施方案中,感兴趣的样品或标记的样品可任选地在与一种或多种可溶胀材料前体接触之前用洗涤剂处理。使用洗涤剂可改善样品的润湿性或破坏样品以允许一种或多种可溶胀单体前体渗透到整个样品中。
在一个实施方案中,感兴趣的样品被一种或多种单体或包含一种或多种单体或前体的溶液渗透,然后根据正在进行的方法的步骤反应以形成可溶胀或不可溶胀的聚合凝胶。例如,如果感兴趣的样品将被包埋在聚丙烯酸钠中,则将包含单体丙烯酸钠和丙烯酰胺以及选自N,N-亚甲基双丙烯酰胺((BIS)、N,N’-(1,2-二羟基乙烯)双丙烯酰胺)和(DHEBA)N,N’-双(丙烯酰基)胱胺(BAC)在整个样品中灌注。
一旦样品或标记的样品被渗透,则将溶液活化以形成聚丙烯酸钠或其共聚物。在一个实施方案中,包含单体的溶液是含水的。
在一个实施方案中,样品(例如标记样品)中的一种或多种蛋白质在扩展之前锚定或交联至可溶胀材料。这可优选地通过用可溶胀材料化学交联可检测标记来完成,诸如在聚合或原位形成可溶胀材料期间或之后。
在一个实施方案中,在标记样品已经锚定到可溶胀材料之后,任选地使样品经受内源生物分子(或感兴趣样品的物理结构,其中样品不是生物材料)的酶、化学和/或物理破坏,使得可检测标记诸如荧光染料分子或抗体完整并且锚定到可溶胀材料。以这种方式,样品-可溶胀材料复合物的机械性能变得更加空间均匀,从而允许具有最小伪影的各向同性扩展。
如本文所使用的,感兴趣样品的术语“消化”或“样品的内源性物理结构的破坏”或术语“内源性生物分子的破坏”可互换使用,并且通常指样品的物理、化学或酶消化、破坏或破裂,使其将不抵抗扩展。
在一个实施方案中,蛋白酶被用于消化样品-可溶胀材料复合物。破坏优选不影响可溶胀材料的结构,但破坏样品的结构。因此,样品破坏应对可溶胀材料基本呈惰性。消化程度可足以危及样品的机械结构的完整性,或者可为完全的使得样品-可溶胀材料复合物基本上不含样品。
在一个实施方案中,样品的物理破坏通过更温和的破坏处理来完成,所述破坏处理使对各个蛋白质的损伤最小化,从而允许在扩展后对蛋白质进行染色和其他处理。在一些实施方案中,这种更温和处理是通过使用LyC进行。在一些实施方案中,这种更温和处理是通过压热样品来进行。
然后各向同性地扩展样品-可溶胀材料复合物。在一个实施方案中,将溶剂或液体加入复合物中,然后被可溶胀材料吸收并引起溶胀。在一个实施方案中,所述液体是水。在可溶胀材料是水可溶胀的情况下,可使用水溶液。
在一个实施方案中,加入水允许包埋的样品在三维中扩展至其原始尺寸至少3倍,优选4倍,优选5倍或更多。因此,样品的体积可增加100倍或更多。这是因为聚合物包埋在整个样品中,因此,随着聚合物溶胀(生长),它也使组织扩展。因此,组织样品本身变大。令人惊讶的是,随着材料各向同性地溶胀,锚定标签保持其相对的空间关系。
具有感兴趣的包埋样品的溶胀材料可在任何光学显微镜上成像,允许低于经典衍射极限的特征的有效成像。由于所得样本优选是透明的,因此可使用能够大体积、广角视野、3D扫描的定制显微镜与扩展样品一起使用。所述方法还提供了可选步骤,包括扩增可检测标记。
如本文所用,术语“ExM工作流程”是指使用可溶胀材料灌注化学固定和透化的感兴趣样品的过程,所述可溶胀材料经历原位聚合(即凝胶化),样品-聚合物复合物的消化和样品-聚合物复合物的扩展。
如本文所用,术语“proExM工作流程”是指将固定样本的蛋白质处理锚定到可溶胀材料(例如通过AcX处理),随后凝胶化、消化、扩展以及成像的过程。
实施例
原料溶液
4%多聚甲醛
4%多聚甲醛(来自Electron Microscopy Science 16%原料)
1x PBS
骤冷溶液(在4C下储存,可在延长时间段内使用)
1x PBS
100mM甘氨酸
蛋白质锚定溶液
1x PBS
0.1mg/mL 6-((丙烯酰基)氨基)己酸,琥珀酰亚胺酯(丙烯酰基-X,SE)
组织破坏溶液(高压灭菌版)
100mM Tris碱
1%十二烷基硫酸钠
5%Triton X-100
组织破坏溶液(磷脂酶版)
0.5x PBS
0.1%Triton X-100
磷脂酶A1(Sigma,L3295)100U/mL
磷脂酶D(Enzo,BML-SE301-0025)500U/mL
抗体染色液(在4C下储存,可使用至少1个月)
1x PBS
0.1%Triton X-100
2%正常驴血清
单体溶液:
*所有浓度为g/100mL,除PBS之外
**9.4/10mL(1.06x),根据需要由引发剂、加速剂和抑制剂(见下文)提供余下的6%体积。
材料和原料溶液储存:
丙烯酸钠有时伴随可变的纯度水平,这可能影响性能。对于购买的每个新鲜瓶子,制备38g/100mL(33wt%)的丙烯酸钠储存液并且检查以确保其在正常室内照明下无色。如果原料有黄色色调(见图1),则从中制成所述原料的瓶子将被丢弃。一旦打开,丙烯酸钠储存在-20℃的密闭、低湿度或干燥器腔室内,因为固体对湿度敏感。APS粉末和100%TEMED溶液储存在室温干燥器中。
将单体溶液在-20℃下混合储存长达1个月。TEMED、APS和H-Tempo原料溶液可保存在-20℃,并且TEMED和APS原料通常至少每2周重新制作一次。
切片凝胶溶液:在冰上混合以下4种溶液。单体溶液+TEMED加速剂+APS引发剂溶液+4-羟基-TEMPO(缩写4HT)抑制剂溶液。需要最后加入引发剂溶液,以防止过早凝胶。需要将溶液进行涡旋以确保完全混合。
每个切片需要~200μl的凝胶溶液。针对200μl凝胶溶液,混合以下:
单体溶液(1.06x)(188μl)(保持在4C以防止过早凝胶):
抑制剂溶液(4μl):4-羟基-TEMPO(0.5%下4HT原料溶液,最终浓度0.01%)(抑制凝胶以能够扩散到脑切片中)。
加速剂溶液(4μl):TEMED(10%下的TEMED原料溶液,最终浓度0.2%(w/w)。(加速APS的自由基生成)。
引发剂溶液(4μl):APS(10%下APS原料,最终浓度0.2%(w/w))。(这引发凝胶过程。这需要最后加入)。
用于脑切片的ExM过程
灌注和切片:基本上与常规组织学相同。
1.使用4%多聚甲醛灌注。将脑固定在4%多聚甲醛(例如,过夜或根据需要)后。
2.通过在4C下在骤冷溶液中温育脑1天将甲醛固定物骤冷。
3.在振动切片机上将脑切片切至希望的厚度。
蛋白质锚定:
1.在1x PBS洗涤脑切片,5分钟。
2.在蛋白质锚定溶液中,在室温下温育至少12小时。
3.在1x PBS洗涤切片,5分钟。
凝胶:
1.确保在与凝胶溶液温育前从脑切片中去除过量的PBS。将切片在Eppendorf管的凝胶液中在4C下温育5分钟,并且然后用新的凝胶溶液替换,再持续25分钟。在4C加入APS后立即使用新鲜制备的凝胶溶液。(确保使用至少100倍过量的单体溶液。例如,每个脑切片~200μl的凝胶溶液。两次温育中的每一次需要~100μl)。
2.将切片从Eppendorf管转移到凝胶室中,并且然后在37C温育2小时。通过将切片夹在滑片和盖玻片之间构成凝胶室(图2),在组织部分任一侧具有间隔物以防止组织切片受压(见下图)。Superfrost滑片(例如VWR 48311-703)可以很好地作为底片起作用。截至100μm的部分,#1.5盖玻片可用于间隔物,并且对于200μm的部分,盖玻片可堆叠两个盖玻片厚度。(通过用钻石划线器切割完整盖玻片很容易制作间隔物。)确保切片平整,并且避免气泡被截留在腔室内。
样本回收:
1.轻轻地去除顶盖玻片和间隔物。
2.使用剃刀刀片修剪/刮掉切片上过量的凝胶。此时,切片/凝胶仍粘附到底部载玻片上。
3.使用1M NaCl洗涤5分钟。
4.用油漆刷轻轻地从滑片上去除标本。此时样本可在1M NaCl中储存数天。
组织破坏(选择一个版):
高压灭菌版
1.在高压灭菌安全容器诸如聚丙烯管中,用组织破坏溶液(高压灭菌版)洗涤2x15分钟。
2.用组织破坏溶液再洗一次。在液体灭菌环境中用高压灭菌处理,峰值温度121C,保持时间60分钟。对于我们的高压灭菌器,这种处理总共花费约2小时。
磷脂酶版:
1.在1x PBS中洗涤2x 15分钟。
2.在组织破坏溶液(磷脂酶版)中在37C下温育样本3天。
抗体染色:
1.将样本移入多壁板,其中孔足够大以便在充分扩展后可容纳它们。
2.用0.1%Triton洗涤2x,15分钟,然后用抗体染色缓冲液洗涤1x,15分钟。
3.如标准免疫染色方案,用稀释到抗体染色缓冲液1:200中或根据需要的一级抗体温育。
4.用抗体染色缓冲液洗涤2x,30分钟。
5.如标准免疫染色方案,用稀释到抗体染色缓冲液1:200中或根据需要的二级抗体温育。
6.用抗体染色缓冲液洗涤2x,30分钟。
7.通过在无盐水中彻底洗涤来扩展,例如,在比初始凝胶体积大200倍的体积的水中洗涤4x 15分钟。
使用常规荧光、共焦显微镜或其他所需观测仪器的图像
如图2a-图3c所示,使用高压灭菌版的组织破坏方案,扩展之前(a)和之后(b),Thy1-YFP-表达大脑组织的落射荧光图像(仅绿色通道)。扩展后共焦图像(c)。扩展组织是使用针对绿色荧光蛋白(GFP,绿色)、GAD65/67(红色)和SV2(蓝色)的一级抗体来抗体染色。比例尺:(a)50um,(b)500um扩展前(2.2mm扩展后),(c)10um扩展前(44um扩展后)。
荧光蛋白筛选(图4a,b)。大部分哺乳动物质粒获自Addgene(表1和3)。为了构建剩余的那些,pmKate2-H2B-N1和pPATagRFP-H2B-N1质粒,将相应的基因作为AgeI/NotI片段进行PCR扩增,并与pH2B-LSSmOrange-N1(Addgene)中的LSSmOrange基因交换。为了产生NLS-iRFP融合蛋白,将编码iRFP基因的PCR扩增的AgeI/NotI片段与pNLS-LSSmKate2-N1(Addgene质粒#31871)中的LSSmKate2基因交换。将HEK293FT(Invitrogen)和HeLa(ATCCCCL-2)细胞在补充有10%FBS(Invitrogen)、1%青霉素/链霉素(Cellgro)和1%丙酮酸钠(BioWhittaker)的DMEM培养基(Cellgro)中培养。使用HEK293FT和HeLa细胞以便于转染,通过STR分析验证细胞系并且通过制造商检查支原体污染。根据制造商的方案使用TransIT-X2转染试剂(Mirus Bio)转染细胞。使用配备有10x NA 0.3物镜,具有390/22nm、438/24nm、475/28nm、510/25nm、585/29nm以及631/28nm激发器(Semrock)的SPECTRA X光引擎(Lumencor)以及由NIS-Elements AR软件控制的5.5Zyla相机(Andor)的Nikon Eclipse Ti倒置显微镜,在转染后24小时进行活HEK293FT细胞的广角成像。活细胞成像后立即将细胞培养物用4%多聚甲醛固定10分钟,并且用0.1%Triton-X透化15分钟,用PBS(Cellgro)洗涤3次5分钟,并且用0.1mg/ml AcX(LifeTechnologies)处理至少6小时,凝胶化并且使用蛋白酶K消化过夜,如下所述(参见“AcX处理”和“凝胶化、消化和扩展”部分)。
消化后,通过用PBS充分洗涤处理样品,并且然后在1M NaCl和60mm MgCl2中收缩(除了对氯化物敏感的YFP20,并且因此在扩展状态下测量)。对于图12所示的对照实验,仅用PBS洗涤凝胶。在MATLAB中执行SIFT/RANSAC算法实现样品处理前和处理后图像的对准。荧光核的通过CellProfiler21的自动Otsu阈值允许在样品处理前后在同一组细胞中自动测量荧光强度。在样品处理前后每个核的强度测量通过分段面积归一化以考虑到荧光团稀释(使用面积,因为落射荧光光学分割减轻了对亮度的轴向扩展效应)。
在ProExM期间荧光染料保持的量化。荧光二级抗体(山羊抗兔,10μg/mL)购自商业供应商(关于荧光二级抗体的列表,参见表2)。如下所述,通过proExM将小鼠皮层成像前后来量化保持(图4c)。用抗Homer一级抗体(Synaptic Systems;参见表4)和表2中描述的不同的二级抗体来对野生型皮质部分(由于Thy1-YFP串扰,仅用于Alexa 488)和Thy1-YFP脑切片(50μm厚)染色。
表4.使用的一级抗体。
凝胶前用4x 0.13NA物镜拍摄脑切片的落射荧光图像。在proExM凝胶化和消化后,用PBS(3x 30分钟)充分洗涤脑切片,并且在相同的成像条件下再次拍摄切片的落射荧光图像。使用皮层中感兴趣的区域来确定proExM处理期间的荧光损失。样品处理之前和之后的强度测量通过分割区域归一化以考虑到荧光团稀释。
结构化照明显微镜扩展前成像。将HeLa细胞用4%多聚甲醛固定10分钟,用PBS洗涤3次,持续5分钟,并且用0.1%Triton-X透化15分钟。使用10μg/mL的浓度的具有0.1%Triton X-100和2%正常驴血清(PBT)的封闭缓冲液1x PBS中的一级抗体(羊抗微管蛋白,Cytoskeleton ATN02)将固定HeLa中的微管染色,持续1-4小时,并且然后在PBS中洗涤3次,每次5分钟。然后将样本与PBT中的二级抗体(驴抗羊,Alexa 488,Life Technologies,10μg/mL)一起温育1-4小时,并且然后在PBS中洗涤三次,持续5分钟。制备50μm脑组织切片并且用一级和二级抗体(兔抗-Tom20,Santa Cruz Biotech sc-11415和羊抗-兔Alexa 568(Life Technologies))染色,如下所述。超分辨率结构化照明显微镜成像在具有100x1.40NA(Olympus)油物镜的Deltavision OMX Blaze(GE healthcare)SIM显微镜上进行。将染色的细胞用SlowFade Gold(Invitrogen)抗衰减试剂进行成像以便抑制光漂白和实现扩展前成像的折射率匹配。
测量误差量化。相同的视野在扩展前和扩展后成像。首先通过旋转、平移和均匀缩放将扩展后图像配准到相应的扩展前图像上。在标本倾斜在扩展前和扩展后成像之间改变,则使用Fiji 3D Viewer软件包使用3D旋转进行校正而不进行缩放。然后将这些缩放后的图像再次配准到扩展前图像中,但这次在Matlab22中使用基于B样条的非刚性配准软件包来捕获扩展过程中的任何不均匀性。使用缩放不变特征变换(SIFT)关键点23自动生成配准控制点。使用VLFeat开放源代码库24生成SIFT关键点,并使用随机样品一致性(RANSAC)来估计限于旋转、平移和缩放的几何变换。将缩放扩展后图像映射到扩展前图像的矢量变形场表示扩展后图像中的每个点相对于理想均匀扩展的偏移。通过减去任意两点处的结果向量,使用扩展后图像来测定相对定位误差,以测量这两点之间的距离。对可能的点对点测量结果的整个群体进行采样,以确定作为测量长度的函数的这些测量的均方根误差。
Brainbow3.0注射液和抗体染色。如先前所述进行Brainbow3.0rAAV(Universityof Pennsylvania,Penn Vector Core)注射13。简而言之,用异氟烷连续麻醉转基因小鼠,并将其头部固定在立体定位仪上。手术在无菌条件下发生,其中动物躺在加热垫上。通过34号注射针以0.2微升/分钟的速率将2μL AAV混合物(7.5x 1012基因组拷贝数/mL)注射到脑(例如皮质、海马体)中,之后使针静置于注射部位5分钟以允许病毒扩散。动物表达病毒3-4周,然后灌注(参见“小鼠灌注”)。
由Cai实验室生产针对Brainbow3.0荧光团的一级抗体(鸡抗GFP,豚鼠抗mKate2,大鼠抗mTFP)。在抗体染色(室温(RT)下的所有温育)之前,用含有0.1%Triton X-100和2%正常驴血清(PBT)的1x PBS将切片透化并封闭30分钟。在PBT中,将切片与一级抗体在4℃温育3天,并且然后用PBT洗涤四次,每次30分钟。在室温下将切片与二级抗体一起温育1天。使用的二级抗体是:山羊抗鸡Alexa 488、山羊抗大鼠Alexa 546(Life Technologies)和驴抗豚鼠CF633(Biotium),均为10μg/mL。
小鼠灌注。以下所有溶液均由1x磷酸盐缓冲盐水(PBS)制成。小鼠用异氟烷麻醉并用冰冷的4%多聚甲醛经心脏灌注。解剖出脑,在4℃下4%多聚甲醛中放置1天,然后移至100mM甘氨酸中。切片(50μm和100μm)在振动切片机(Leica VT1000S)上切片并且在4℃下储存直到染色。
AcX处理。将丙烯酰基-X,SE(6-((丙烯酰基)氨基)己酸,琥珀酰亚胺酯,在此缩写为AcX;Thermo-Fisher)以10mg/mL的浓度重悬于无水DMSO中,等分并且冷冻储存于干燥环境。用这种方法制备的AcX可储存长达2个月。为了锚定,将细胞和组织切片在以0.1mg/mL的浓度稀释于PBS中的AcX中在室温下温育>6小时。对于厚的组织(>100微米),AcX渗透深度和标记均匀性可通过将样品在较低pH下,在较低温度下和基于2-(N-吗啉代)乙磺酸(MES)的盐水(100mM MES,150mM NaCl;图16)中改善。组织切片可以在摇动器或摇杆上温育以确保反应期间混合。
凝胶化、消化和扩展。对于AcX锚定的荧光蛋白和抗体染色,可以按照先前所述进行以下步骤-凝胶化、消化和扩增1。简而言之,将单体溶液(1x PBS,2M NaCl,8.625%(w/w)丙烯酸钠、2.5%(w/w)丙烯酰胺,0.15%(w/w)N,N'-亚甲基双丙烯酰胺)混合,在等分试样中冷冻并且在使用前解冻。使用前将单体溶液冷却至4℃。将浓缩原料物(10%w/w)过硫酸铵(APS)引发剂和四甲基乙二胺(TEMED)加速剂分别加入到单体溶液中至多0.2%(w/w)。对于切片,从0.5%(w/w)原料加入至多0.01%(w/w)的抑制剂4-羟基-2,2,6,6-四甲基哌啶-1-氧基(4-羟基-TEMPO)以在单体溶液扩散到组织部分期间抑制凝胶化。将细胞或组织切片与单体溶液加上APS/TEMED(和用于切片的4-羟基-TEMPO)在4℃下,分别针对培养细胞和脑切片来温育1分钟、30分钟,并且然后转移到加湿的37℃的培养箱中2小时以用于凝胶化。
在消化缓冲液(50mM Tris(pH 8),1mM EDTA,0.5%Triton X-100,1M NaCl)中将蛋白酶K(New England Biolabs)以1:100稀释至8单位/毫升,并且与充分浸入蛋白酶溶液中的凝胶在室温下温育过夜(此步骤也可以在37℃下进行4小时)。接下来将消化的凝胶置于过量体积的双重去离子水中0.25-2小时以扩展,更厚的凝胶需要更长的时间。此步骤在淡水中重复3-5次,直到扩展样品的尺寸达到稳定。
扩展后荧光显微术。在具有60x 1.40NA油物镜的Andor旋转盘(CSU-X1Yokogawa)共焦系统上进行细胞的扩展后共焦成像(图4)。为了量化比较之前对之后的组织切片和低放大倍数的扩展系数,将样品在具有4x 0.13NA空气物镜的Nikon Ti-E落射荧光显微镜上进行ExM前成像(图5a-d、图8a-b、图10b、图12a-g以及图15a和15b)。对于图6a-b,使组织切片在具有10x 0.45NA的Nikon Ti-E落射荧光显微镜上成像。除此之外,除了图8、图10a、图11以及图13以外,使用具有40x 1.15NA水浸物镜(Nikon)的Andor旋转盘(CSU-X1Yokogawa)共焦系统使存在的所有其他组织成像。针对图16i,使用具有10x 0.3NA空气镜片的ZeissLSM 710。
为了在扩展后成像期间稳定凝胶以防止漂移,将凝胶放置在玻璃底6孔板中,其中去除所有过量的液体。如果固定需要的话,将液体低熔点琼脂糖(2%w/w)吸移在凝胶周围并且允许固化,以在成像之前包封凝胶。
PALM成像。使用Olympus 1.4NA PLAPON 60x OSC物镜和定制管透镜(LAO-300.0,Melles Griot),在定制的三相机RAMM帧显微镜(ASI)上记录PALM数据,从而产生100x的总放大倍数25。将2mm厚的四波长激发二向色性(ZT405/488/561/640rpc,Chroma),2mm厚的发射二向色性(T560lpxr,Chroma)和带通发射过滤器(FF01-609/54-25,Semrock)过滤发射的光。Dendra2通过每200ms的405nm(50W/cm2)的100μs长激发脉冲进行光转换,在图像采集期间每200ms斜率上升至1.2ms长脉冲。在2kW/cm2的估计样品功率水平下,使用NI-DAQ-USB-6363采集板(National Instruments)实现Stradus 405-100激光器(Vortran)的频闪405-nm激发,使用555-nm DPSS激光器(CrystaLaser)激发经光转换的Dendra2分子。在20帧/秒下,使用具有背照式EMCCD相机(Andor Technology,Ixon Ultra DU-897_BV,17MHz EM扩增器,Gain 500,全芯片)的μManager(v.1.4.20)26检测荧光。
颗粒定位。定位器27用于8路邻接颗粒检测,具有20个GLRT敏感性和1.3个像素的PSF。使用专门基准标记物对所得颗粒进行漂移校正。对于每个检测到的颗粒,使用IgorPro版本6.36中编写的分析程序将整合荧光强度转换为光子计数。平均和中位定位误差使用参考文献28中的方程6确定。
不同组织类型的ProExM。使用冷丙酮后固定的胰腺、脾和肺的小鼠正常新鲜冷冻组织部分的标准组织学制备物(5μm-10μm)从US Biomax(分别MOFTS036、MOFTS051和MOFTS031)获得。在抗体染色之前,用具有0.1%Triton X-100和2%正常驴血清(PBT)的1xPBS将组织封闭30分钟。组织在室温下用一级鸡抗波形蛋白(Abcam)染色4小时,并且然后使用PBT洗涤四次,30分钟。在室温下将切片与二级抗体温育2小时(抗-鸡Alexa 488,LifeTechnologies)。如上所述,进行扩展前成像。在上述(不同之处在于在60℃下进行消化4小时)凝胶化、消化和扩展前,在室温下将组织与PBS中的0.05mg/mL AcX温育过夜。
内源性蛋白质的抗体染色。在凝胶化之前或高压灭菌或LysC处理之后,将样本在具有0.1%Triton X-100和2%正常驴血清(PBT)的1x PBS中在室温(RT)下温育2小时以封闭,并且在凝胶前样本的情况下加以透化。标本与在PBT中3μg/mL的一级抗体温育4小时(RT),并且然后用PBT洗涤四次,30分钟。标本与在PBT中20μg/mL的二级抗体温育4小时(RT),并且然后用PBT洗涤四次,至少30分钟。使用的二级抗体为:山羊抗-鸡Alexa 488(Life Technologies)、山羊抗-兔Alexa 546(Life Technologies)和山羊抗-小鼠CF633(Biotium),不同的是山羊抗-鸡Alexa 546(Life Technologies)用于图8e、g(ii)、h(ii),并且山羊抗-兔Alexa 488(Life Technologies)用于图4e。
使用高压灭菌的样本破坏。凝胶化后,凝胶从凝胶腔室中回收并且在1M NaCl中洗涤。将凝胶在破坏缓冲液(100mM Tris碱,5%Triton X-100,1%SDS)中洗涤15分钟,然后置于新鲜的破坏缓冲液中,并且在121℃温度的液体灭菌模式下用高压灭菌处理1小时。这种处理必须在高压灭菌安全的容器诸如聚丙烯管中进行。然后将凝胶转移至孔板以用于抗体染色和成像,并且在PBT(1xPBS,2%正常驴血清,0.1%Triton X-100)中洗涤以去除破坏缓冲液。
用LysC温和消化。凝胶化后,将凝胶在具有600U/ml II型胶原酶(LifeTechnologies)的HBSS缓冲液(具有钙和镁,ThermoFisher Scientific)中在37℃下预处理2-4小时。然后将凝胶在LysC消化缓冲液(25mM Tris-HCl,1mM EDTA,pH 8.5)中洗涤5分钟,并且在37℃下与33μg/ml LysC(Promega)温育至少8小时。最后,将凝胶在LysC消化缓冲液3x中洗涤,各自30分钟,并且使用上述相同的步骤经受免疫染色。
SNOTRAP-生物素的合成。向干燥DMF(5mL)中的搅拌的2-(二苯基膦基)-苯硫醇(100mg,0.34mmol)连续加入生物素-PEG3-丙酸(100mg,0.22mmol,ChemPep公司)、N,N’-二环己基碳二亚胺(DCC)(70mg,0.34mmol)和二甲基氨基吡啶(DMAP)(4mg,0.03mmol)。将所得混合物在室温下搅拌7小时,并且然后将所得澄清溶液在减压下浓缩并且通过快速色谱法(己烷/EtOAc/MeOH梯度)纯化以给出所需产物(产率30%)。将SNOTRAP探针在具有254nm光电二极管阵列UV检测器的1100HPLC系统(Agilent Technologies,Wilmington,DE)上再纯化。HPLC柱和溶剂体系如下:用水(A)和乙腈(B)中的0.1%甲酸的线性梯度洗脱半制备Phenomenex Luna C18(25cm x 9.4mm,10μm)柱,流速为2.5毫升/分钟。溶剂组合物最初为40%,5分钟,70%,10分钟,90%,20分钟,并且然后在8分钟内进一步至95%B。1H NMR(500MHz,CD3CN,δ)7.42-7.38(m,9H),7.23–7.18(m,4H),7.84(m,1H),4.60-4.51(m,2H),3.67-3.51(m,12H),3.2(m,3H),2.8(m,2H),2.55(t,2H),2.15(t,2H),1.57-3.51(m,6H);13CNMR(125MHz,CD3CN,δ)199.19,172.5,164.5,144.8,138.1,137.0,134.8,129.9,129.6,129.6,118.3,69.2,63.1,62.3,45.9,42.5,38.2,27.1,23.1,22.5;31P NMR(202MHz,CD3CN,δ)-10.3;HRMS-ESI+(m/z):[M+H+]+,针对C37H47N3O6PS2计算,724.2638;发现724.2632。
SNOTRAP染色的ProExM。对于SNOTRAP染色,使用PBS将一级神经元培养物洗涤3X 5分钟并且使用冷甲醇在-20℃固定15分钟。将神经元与PBS-Triton X100(0.3%v/v)中的300nM N-乙基马来酰亚胺(NEM)(Thermo Scientific)在37℃下温育30分钟以封闭蛋白质上的游离-SH基团。然后用PBS将细胞洗涤3X 5分钟,并且与乙腈-PBS-triton(50%:50%v/v)中的SNOTRAP探针(250uM)在室温下温育1小时,并且然后进一步用1/500稀释(PBS-Triton)的链霉亲和素-Alexa 488(Thermo Scientific)在室温下温育1小时,并且然后洗涤5X5分钟。如上所述进行抗微管蛋白(Alexa 546二级)和proExM的抗体染色。
动物护理。动物护理和使用的所有方法均由麻省理工学院动物护理委员会批准,并符合美国国家卫生研究院关于护理和使用实验室动物的指南。本研究使用一只体重12kg的成年雄性恒河猴(Macaca mulatta),以及1只C57BL/6小鼠、4只Emx1-Cre小鼠和10只~1-3月龄的Thy1-YFP小鼠。使用小鼠时不考虑性别。
恒河猴程序。使用立体定向坐标以七氟醚麻醉进行病毒注射以靶向8个注射部位。将病毒(AAV8,)离心并且加载到已用硅油(Sigma)回填的10μL气密注射器(Hamilton)中。使用立体定向显微操作臂(David Kopf Instruments)和UMP3微型注射器喷射泵(WorldPrecision Instruments)将总共3μL病毒以100nL/分钟-200nL/分钟的速率在两个位置处灌注到脑中(深,然后500μm浅表)。每次注射后,取出前将针头和注射器放置10分钟。所有注射均使用钝的33G针头。还给予1mg地塞米松以防止脑肿胀。病毒注射后4周发生安乐死。在用磷酸盐缓冲盐水(PBS)和4%多聚甲醛(PFA)灌注之前施用过量的戊巴比妥。然后提取脑、封闭并且储存在具有0.1%叠氮钠溶液的20%甘油中,并且最后切成40μm切片机部分。
虽然已经具体显示并且参考其优选实施方案描述了本发明,但是本领域技术人员会理解可以在形式和细节中作多种改变而不脱离由附加的权利要求涵盖的本发明的范畴。
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Claims (14)
1.一种用于感兴趣的样品的蛋白质的保持和成像的方法,其包括以下步骤:
(a)使所述样品内的蛋白质与双官能交联剂缀合;
(b)将所述样品包埋在可溶胀材料中,其中所述样品内的蛋白质锚定到所述可溶胀材料;
(c)使所述样品经受消化;
(d)使所述可溶胀材料溶胀以形成扩展样品;以及
(e)对所述扩展样品成像。
2.根据权利要求1所述的方法,其中所述双官能交联剂包含蛋白质反应性化学基团和凝胶反应性化学基团。
3.根据权利要求2所述的方法,其中所述双官能交联剂是6-((丙烯酰基)氨基)己酸的琥珀酰亚胺酯(AcX)。
4.根据权利要求1所述的方法,其中所述消化是所述样品的物理、化学或酶破坏。
5.根据权利要求1所述的方法,还包含所述样品的抗体染色的步骤。
6.根据权利要求5所述的方法,其中在与所述双官能交联剂缀合的处理之前,所述样品用一种或多种抗体染色。
7.根据权利要求5所述的方法,其中在所述样品处于扩展状态后,所述样品用一种或多种抗体染色,并且所述破坏方法保存蛋白质抗原性。
8.根据权利要求1所述的方法,其中所述样品表达一种或多种荧光蛋白。
9.根据权利要求8所述的方法,其中所述一种或多种荧光蛋白锚定到所述可溶胀材料。
10.根据权利要求1所述的方法,其中所述样品通过加入水以溶胀所述可溶胀材料来各向同性地扩展。
11.根据权利要求4所述的方法,其中所述消化步骤包括使用LysC、高压灭菌或蛋白酶K来处理所述样品。
12.根据权利要求4所述的方法,其中所述破坏法是酶消化。
13.根据权利要求1所述的方法,其中将所述生物样品包埋在可溶胀材料中包括使用包含可溶胀聚合物的前体的组合物渗透所述生物样品并且原位形成可溶胀聚合物。
14.根据权利要求1所述的方法,其中所述至少一种蛋白质锚定到所述可溶胀材料。
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