High-Resolution Intravital Microscopy
High-Resolution Intravital Microscopy
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DOI：
10.1371/journal.pone.0050915
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发表时间：
2012-12-14
期刊：
影响因子：
3.7
通讯作者：
Niesner, Raluca
中科院分区：
文献类型：
--
作者：
Andresen, Volker;Pollok, Karolin;Niesner, Raluca
细胞通讯构成了生命的基本机制，例如通过允许通过神经系统中的突触传递信息以及通过在免疫应答过程中导致细胞活化。监测活体成年生物体内细胞与细胞的相互作用对于得出有关细胞、组织和器官命运的行为的结论至关重要。到目前为止，还没有可用的技术能够以亚细胞分辨率在活的成年生物体的组织深处进行动态成像，即在很少的蛋白质分子水平上进行检测。在这里，我们提出了一种新的方法，称为多光束条纹照明第一次适用于结构照明的原理和优点，空间调制的激发模式，激光扫描显微镜。我们在双光子显微镜中使用这种方法-最合适的光学深层组织成像技术。与标准双光子显微镜相比，它实现了显着的对比度增强和高达3倍的轴向分辨率（光学切片），而光漂白，光损伤和采集速度是相似的。其成像深度与多焦双光子显微镜相当，仅略低于标准单光束双光子显微镜。准确地说，我们在小鼠淋巴结内的研究表明，在表面以下80 mm的深度处，轴向分辨率提高了216%，横向分辨率提高了23%。因此，我们是第一次能够可视化的动态之间的相互作用B细胞和免疫复合物沉积在滤泡树突状细胞内的生发中心（GC）的活小鼠。这些相互作用在克隆选择过程中起决定性作用，导致体液免疫应答的亲和力成熟。这种新的高分辨率活体显微镜方法在神经科学，免疫学，癌症研究和发育生物学的众多应用中具有巨大的潜力。此外，我们的条纹照明的方法是能够提高分辨率的任何激光扫描显微镜，包括共焦显微镜，通过简单地选择一个合适的检测器。引用：Andresen V，Pollok K，Rinnenthal J-L，Oehme L，Gunther R等（2012）高分辨率活体显微镜。PLoS ONE 7（12）：e50915. doi：10.1371/journal. pone。0050915
Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped- illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 mm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped- illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector. Citation: Andresen V, Pollok K, Rinnenthal J-L, Oehme L, Gunther R, et al. (2012) High-Resolution Intravital Microscopy. PLoS ONE 7(12): e50915. doi:10.1371/journal.pone. 0050915