Summary

光片为主的生活的荧光显微镜或固定和染色<em>赤拟谷盗</em>胚胎

Published: April 28, 2017
doi:

Summary

成像昆虫胚胎与基于光的片材的荧光显微镜的形态已成为现有技术的状态。该协议概述并比较适合赤拟谷盗胚胎三个安装技术中,引入了两个新的定制的转基因系非常适合于实时成像,讨论必需的质量控制和指示当前实验的限制。

Abstract

在赤拟谷盗赤拟谷盗已成为发育遗传学及进化发育生物学的一个重要昆虫的模式生物。 胚胎与基于光的片材的荧光显微镜观察具有优于常规宽场和共聚焦荧光显微镜多个优点。由于基于光的片材显微镜的独特性质,活标本的三维图像可以记录具有高信号噪声比和比上几个周期显著降低光致漂白,以及沿多个方向的光毒性天。随着四年多的发展方法和数据的不断增加,时间似乎是适当的建立光片技术在社区的使用以及在昆虫社会大众的标准作业程序。这个协议描述了适合˚F三个安装技术或不同的目的,提出了两种新的定制的转基因线适合于长期实时成像,表明5个的荧光染料来标记固定胚胎的细胞内结构以及用于记录数据的及时评估提供了关于数据的后处理的信息。代表性的结果集中在长期实时成像,光学切片并沿多个方向相同的胚胎的观察。相应的数据集提供的可下载资源。最后,协议讨论了用于实时成像测定,电流限制和所概述的程序到其它昆虫物种的适用性的质量控制。

该协议主要针对谁寻求成像解决方案,超越标准的实验室设备发育生物学家。它促进了连续尝试关闭技术上导向的实验室/社区,其发展和完善万分之一之间的差距复制方法论,生命科学实验室/社区,这需要技术上的挑战“插件和播放的解决方案。此外,它支持移动的生物学问题成为人们关注的焦点的公理化方法。

Introduction

在赤拟谷盗赤拟谷盗 ,属于大家族暗中甲虫(拟步甲科),具有农业和生命科学领域内历史悠久,是果后的第二个最好的研究模型昆虫的模式生物飞行果蝇 。在过去的四个十年里,它成为发育遗传学的强大和受欢迎的昆虫的模式生物,在进化发育生物学,并在过去的二十年中,在各种原因的胚胎形态:

果蝇同属Holometabola,但约300万年前的1,2,3,4分歧。而果蝇胚胎发育通常被认为是高度衍生, 示出devel的更祖模式即在昆虫物种5,6,7,8,9的一个相当大的比例发现opment。首先, 呈现出非渐开头的发展, 即,其口器和天线胚胎发生10,11,12,13,14,15中已出现。其次, 如下短胚芽发展, 腹部段germband伸长16,17,18,19在从后部生长区依次加入的原理。第三, 开发和后来降级两个额外的胚胎外膜羊膜,它涵盖了胚胎只腹侧,和浆膜,其完全包住胚胎20,21,22。两种膜起到至关重要的形态发生23以及抗微生物24,2526干燥保护作用。第四,embryonically显影腿是在幼虫生命阶段完全功能和蛹变态27,28,29,30,31时用作原基为成人腿。

由于其体积小,适度的要求,在实验室的培养是相当简单的。野生型培养物(WT)菌株或转基因品系通常由周围100-300成人和可以保持一升玻璃瓶(足迹80厘米2)往三四十厘米高(约50克)与由全谷物小麦的生长培养基内面粉补充有非活性干酵母。供水是没有必要的。这样,即使小型实验室,以保持小型或中型商用虫孵化器内的几十个甲虫文化。 的后面发育阶段(幼虫后约4龄,蛹和成虫)很容易从生长培养基通过筛分分离。同步胚胎,恒温产蛋介质上短期内得到成年人。对于快速发展,甲虫培养物保持在32℃(每代约四周),而贮存保持在22-25℃通常执行(每一代约十周)。

在过去的十年中,许多标准TEChniques已经逐渐适应和优化,总结在新兴的模式生物32。非常重要的是先进遗传方法例如胚胎33,幼虫34,35或亲本36,37基于RNA干扰的基因敲除,与任一piggyBac转 38,39米诺斯 40转座系统和CRISPR /基于Cas9基因组种系转化工程41。此外, 基因组被测序大约十年前42,并且现在是在第三轮基因组的装配释放43,其允许高效且全基因组鉴定和基因44的系统的分析</s向上>或其它遗传元件45,46。另外,四个其它鞘翅目物种的基因组可用于比较遗传方法47,48,49,50。与测序基因组关联,两次大规模的遗传分析已经执行, ,插入诱变屏幕51和基于干扰系统的RNA基因敲除画面52,53。

与宽视场,共焦或基于光的片材显微镜(LSFM)荧光实时成像允许观察的胚胎形态作为时间多维上下文( 表1)的函数( ,形态发生)。在宽视场和共聚焦荧光显微镜,该EXCIT通货膨胀和发射光通过相同的物镜引导。在这两种方法中,整个试样被照亮每记录二维平面。因此,试样承受非常高的能量水平。在LSFM,只有在焦平面中的荧光团是兴奋由于通过使用两个垂直布置的物镜( 图1)照明和探测的去耦。 LSFM有两种法服实现-该单个平面照明显微镜(SPIM)和数字扫描的激光基于片材的荧光显微镜(DSLM, 图2) -和在传统的方法提供了几个重要的优点:(ⅰ)固有光学切片能力, (ⅱ)良好的轴向分辨率,(ⅲ)强烈降低光致漂白的水平,(ⅳ)非常低的光毒性,(v)的高信噪比,(ⅵ)相对高的采集速度,(ⅶ)成像沿多个方向和(viⅱ)更深的组织穿透由于低数值孔径照射物镜54,55,56的使用。

LSFM已经在已成功地应用于记录几乎整个胚胎的形态发生57和分析外胚膜破裂的原则,在背封23的开端。为了提高LSFM的吸引力在社区和一般昆虫科学,这是非常重要的,建立标准作业程序,改进方法,协议和资源的水平池在显微镜变成了一个易于在发育生物学实验室-use标准工具和生物学问题留在人们关注的焦点。

该协议开始于的基本</ em>的培养, 维修,繁殖和胚胎收集。接着,两个实验策略中示出:(ⅰ)的定制的转基因品系实时成像和(ii)该染色用荧光染料( 表2)固定的胚胎的成像。 (i)所述琼脂糖柱,(ii)所述琼脂糖半球和(iii)所述新的蛛网持有人:随后,具有稍微不同的目的三个安装技术详细( 图3表3)进行说明。该协议然后解释与LSFM获取数据的步骤。影像学技术和关键因素进行了概述。最后,胚胎检索解释并提供了基本的数据处理建议。在代表性结果,从活体成像数据的两个新的定制和胶质-蓝58个的转基因株系被示出和各个成像数据集作为可下载资源提供。此外,图像该染色用各种荧光染料的固定胚胎的数据表示。讨论集中在质量控制方面,实时成像方式的电流限制和协议的其他物种的适应性。

该协议是针对配有样品室和用于标准化样品架54,59,60,这是典型的直径由金属,塑料或玻璃筒形元件可旋转的夹持机构基于光的片材荧光显微镜写入在毫米范围内。该协议也适用于这两种法服实施方式中, SPIM和DSLM,以及用于与两个或更多照明和检测臂61,62,63设置。代表结果表明在两个光谱信道的数据,绿色(ILlumination用488nm的激光,检测通过五十零分之五百二十五通过七十零分之六百零七带通滤波器的带通滤波器)和红色(照明用561纳米的激光,检测),但是协议可以扩展到三个或四个光谱信道。

Protocol

1. 赤培养的畜牧注意:标准条件定义为在25℃和70%相对湿度在12个小时亮/ 12小时黑暗周期的培养温度。有关赤畜牧业的更多信息,相应的指南可64。此协议需要两个不同的面粉媒体​​,可在千克数量制备和储存数月。 之前用面粉和非活性干酵母工作,存储未开启的包装,在-20℃下放置24个小时,然后让它们升温至室温。这个过程消除?…

Representative Results

这个协议描述了用于生活的荧光成像或固定和染色赤胚胎LSFM实验框架。由于光漂白和光毒性,其光学切片能力的直接结果的低水平,LSFM是特别适合于长期实时成像。 新颖AGOC {ATub'H2B-mEmerald}#1转基因线下的α-微管蛋白1启动子74的控制表达histone2B-mEmerald融合蛋白。它示出了一个增强子捕获状表?…

Discussion

品质管制

在活体成像试验中,准备和记录程序必须是非侵入性的, 即,既不机械和化学处理(收集,dechorionation,安装到样本保持器),也没有观察时的集成能量负载应该影响样品的可行性。对于研究表征WT发展,它是从其中胚生存的录制过程中,成功地检索并发展成一个健康的成年人实验建议只使用数据。成人不应该表现出任何形态畸变,宜肥沃,且其…

Declarações

The authors have nothing to disclose.

Acknowledgements

我们感谢斯文·普拉斯的技术支持。神经胶质蓝色的转基因品系是从格雷戈尔·布彻(哥廷根,德国)惠赠。这项研究是由卓越法兰克福的集群资助上午主在歌德的布赫曼研究所分子生命科学(BMLS,主管恩里科·施莱夫)部分授予EHKS大分子复合物(CEF-MC,EXC 115,扬声器沃尔克·多茨奇)法兰克福大学是由德意志研究联合会(DFG)为主。

Materials

full grain wheat flour Demeter e.V. 1.13E+08 US: whole wheat flour, UK: whole meal flour
405 fine wheat flour Demeter e.V. 1.13E+08 US: pastry flour, UK: soft flour
inactive dry yeast Flystuff / Genesee Scientific 62-106
phosphate-buffered saline (PBS), pH 7.4 Thermo Fisher Scientific 10010-023
sodium hypochlorite, ~12% active Cl Sigma Aldrich 425044-250ML Caution: sodium hypochlorite is corrosive
low-melt agarose Carl Roth 6351.2
6-well plate Orange Scientific 4430500
24-well plate Orange Scientific 4430300
glass capillaries, internal Ø 0.46 mm Brand GmbH + Co KG 7087 09
SYTOX Green Thermo Fisher Scientific 57020 Staining solution preparation is explained in Table 2
YOYO-1 Iodide Thermo Fisher Scientific Y3601 Staining solution preparation is explained in Table 2
BOBO-3 Iodide Thermo Fisher Scientific B3586 Staining solution preparation is explained in Table 2
Alexa Fluor 488 Phalloidin Thermo Fisher Scientific A12379 Staining solution preparation is explained in Table 2
Alexa Fluor 546 Phalloidin Thermo Fisher Scientific A22283 Staining solution preparation is explained in Table 2
sieve, 800 µm mesh size VWR International 200.025.222-051
sieve, 710 µm mesh size VWR International 200.025.222-050 for growth medium preparation (step 1.1)
sieve, 300 µm mesh size VWR International 200.025.222-040
sieve, 250 µm mesh size VWR International 200.025.222-038 for egg laying medium preparation (step 1.2)
glass dish, Ø 100 mm × 20 mm Sigma Aldrich CLS70165102
cell strainer, 100 µm mesh size BD Biosciences 352360
paint brush, head Ø 2 mm VWR International 149-2121
syringe, 1.0 ml B. Braun Medical AG 9166017V
scintillation vials Sigma Aldrich M1152-1000EA
paraformaldehyde Sigma Aldrich 158127 Caution: paraformaldehyde is toxic and corrosive
n-heptane ≥ 99% Carl Roth 8654.1 Caution: n-heptane is flammable and toxic
Triton X-100 Sigma Aldrich X100-100ML Caution: Trition X-100 is corrosive

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Strobl, F., Klees, S., Stelzer, E. H. K. Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos. J. Vis. Exp. (122), e55629, doi:10.3791/55629 (2017).

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