Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.
Three dimensional multiphoton imaging of fresh and whole mount developing mouse mammary glands.
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DOI：
10.1186/1471-2407-13-373
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发表时间：
2013-08-06
期刊：
影响因子：
3.8
通讯作者：
Mueller SC
中科院分区：
文献类型：
--
作者：
Johnson MD;Mueller SC
多光子显微镜在基础和临床研究中的应用越来越广泛，它是对组织细胞表层共聚焦成像的补充。然而，活体组织的光散射特性使得活体组织成像变得困难，并且这在小鼠乳腺中非常明显，所述小鼠乳腺包含填充有围绕导管上皮的脂肪细胞的基质。用胭脂红明矾染色的整个封片乳腺易于存档以供以后参考，并且易于使用明视野显微镜观察以观察导管网络的分支结构。在这里，我们报告的优势，多光子成像的整个安装乳腺。其中最主要的是，末端芽（TEB）和导管上皮的光学切片允许评价结构异常，这是非常难以确定的，无论是使用明亮的视野成像的染色腺体或常规方法的苏木精和伊红染色的固定和石蜡包埋切片。第二个优点是由二次谐波产生（SHG）提供的细节，其中可以观察到胶原纤维的取向和丰度。使用Zeiss LSM 510/Meta/NLO多光子显微镜对GFP-小鼠乳腺进行活体成像或在整个封片制备后进行成像，目的是获得具有3D内容的高分辨率图像，并评估由整个封片制备诱导的任何结构改变。我们描述了一种简单的方法，使用商业共聚焦/多光子显微镜配备了钛-蓝宝石激光器，同时图像胭脂红明矾荧光和胶原纤维网络的SHG与激光激发设置为860 nm。比较固定、染色和整体包埋制备前后相同的终末芽（TEB），并测定胶原网络结构和TEB形态。使用Meta检测器进行光谱发射扫描，探索了激发和发射滤光片的灵活性。使用具有最大孔径的常规共焦检测器检测后向散射或反射的SHG（SHG-B），并且使用非去扫描检测器检测前向散射或透射的SHG（SHG-F）。我们在这里显示，发育中的乳腺被包裹在一层薄而致密的胶原纤维中。稀疏的胶原蛋白层也散布在TEB周围的脂肪细胞基质层之间。在边缘，TEBs的方法外胶原层，但不穿透it. Abnormal乳腺HAI-1转基因FVB小鼠模型被发现含有TEBs与异常口袋的细胞形成额外的管腔和连续的侧芽形成区散布稀疏的胶原纤维。评价了影响实时成像的参数以及固定的未染色和胭脂红明矾染色的完整封片的成像。在活组织中，由来自上皮细胞的GFP和胭脂红明矾信号的光散射诱导的伪影被识别为主要是由于脂肪细胞，在整个安装组织中，由于上皮细胞的密集胭脂红明矾染色。在激发波长750 - 950 nm处检测到胭脂红明矾自发荧光，发射峰位于623 nm（约602-656 nm）。胭脂红明矾荧光的图像在565-615 nm与650-710 nm的发射波长处显著不同。在后者中，主要是上皮（核）可见胭脂红明矾占主导地位。具有495 nm峰值发射的自体荧光源自固定和处理的组织本身，因为其存在于未染色的整个封片中。自发荧光对图像的贡献随着激光激发波长的增加而减小。SHG-B与SHG-F信号显示胶原纤维，并且可以在单个纤维中或在同一层内的不同纤维中发现。这些差异可能反映了胶原纤维成熟的不同状态。从层到层的SHG信号的损失可以归因于由来自致密TEB结构的光散射呈现的伪影，并且除非选择带通发射，否则包含未过滤的非SHG荧光和自发荧光发射。使用光谱发射成像可以增加成像的灵活性，以优化发射带宽并分离SHG-B，GFP和胭脂红明矾信号，尽管传统的滤波器也是有用的。胶原纤维排列和TEB结构在整个安装过程中得到很好的保留，并且通过提取脂肪显著减少了光散射，从而改善了3D结构，特别是对于源自胶原的SHG信号。除了提供明亮的信号之外，胭脂红明矾染色的整个封片载玻片可以回顾性地成像，例如对HAI-1小鼠腺体进行成像，从而揭示异常TEB形态的新方面。这些研究证明了在发育中的乳腺中，与正常和异常TEBS相邻的胶原纤维紧密接触，但相对稀疏，并且能够获得这些高分辨率细节，但受到所讨论的限制。我们的研究表明，TEB架构在处理后基本上没有变化。
The applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed. GFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector. We show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers. Parameters influencing live imaging and imaging of fixed unstained and Carmine Alum stained whole mounts were evaluated. Artifacts induced by light scattering of GFP and Carmine Alum signals from epithelial cells were identified in live tissue as primarily due to fat cells and in whole mount tissue as due to dense Carmine Alum staining of epithelium. Carmine Alum autofluorescence was detected at excitation wavelengths from 750 to 950 nm with a peak of emission at 623 nm (~602-656 nm). Images of Carmine Alum fluorescence differed dramatically at emission wavelengths of 565–615 nm versus 650–710 nm. In the latter, a mostly epithelial (nuclear) visualization of Carmine Alum predominates. Autofluorescence with a peak emission of 495 nm was derived from the fixed and processed tissue itself as it was present in the unstained whole mount. Contribution of autofluorescence to the image decreases with increasing laser excitation wavelengths. SHG-B versus SHG-F signals revealed collagen fibers and could be found within single fibers, or in different fibers within the same layer. These differences presumably reflected different states of collagen fiber maturation. Loss of SHG signals from layer to layer could be ascribed to artifacts rendered by light scattering from the dense TEB structures, and unless bandpass emissions were selected, contained unfiltered non-SHG fluorescence and autofluorescent emissions. Flexibility in imaging can be increased using spectral emission imaging to optimize emission bandwidths and to separate SHG-B, GFP, and Carmine Alum signals, although conventional filters were also useful. Collagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.
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