Three-Dimensional Superresolution Imaging in Living Cells Using Single-Molecule A

描述（由申请人提供）：具有单分子主动控制的活细胞中的3-D超分辨率成像单分子显微成像技术的最新进展已经导致了超分辨率信息，即观察分辨率超过标准衍射极限的物体的能力。这些方法涉及宽场成像，并且需要对分子进行主动控制，以便打开发射器或关闭发射器，从而保持发射器的浓度足够低以抑制单个分子的点扩散函数。通过多次成像、光漂白和再活化循环，获得了超分辨率图像，但仅用于实际三维样品的二维投影。这些方法可以统称为单分子主动控制显微术（SMACM），并且先前主要应用于固定的细胞。然而，许多生物医学感兴趣的样品，如细胞，足够厚，二维成像是一个严重的限制。该研究计划的主要目标是使用SMACM在活细胞中实现三维超分辨率成像。本研究将从三个方面解决三维超分辨成像问题。首先，用于实现主动控制的光学照明将在其强度方面作为时间的函数进行定制，以便增加再激活和成像过程的效率，并最终能够观察时间依赖性变化。其次，显微镜将重新设计，以利用旋转点扩散功能。这依赖于迫使单个发射器的图像在检测器处具有针对样本中的单个分子的不同z位置旋转的形状。这样做的效果是能够更精确地确定样品中各种单分子标记的z位置，当与x-y平面中的精确定位相结合时，将产生超出衍射极限的三维图像信息。第三，除了旋转点扩散函数外，该研究还将实现多平面成像，这将使在更大深度上采集样品的3D信息成为可能。这项研究的结果将使一种新型的细胞光学显微镜成为可能，在这种显微镜下，可以以非侵入性的方式获得关于细胞亚结构（包括单分子）的三维超分辨率信息。单个荧光团作为纳米级光源的功率将被最大限度地利用。通过提供活细胞三维高分辨率光学成像的新方法，这项研究将直接影响生物技术和生物医学应用，因为这些领域目前在许多诊断情况下利用细胞的光学荧光显微镜。目前的趋势是推动越来越小的空间尺度，用于分析单个细胞结构的行为和形态。特异性和非侵入性地分析细胞器和其他微小细胞结构的突变或毒性行为的能力将允许精确评估靶向药物治疗的效用，这将有助于推动疾病点上的医疗干预的未来。

DESCRIPTION (provided by applicant): 3-D Superresolution Imaging in Living Cells with Single-Molecule Active Control Recent advances in microscopic imaging techniques with single molecules have led to superresolution information, that is, the ability to observe objects with resolution beyond the standard diffraction limit. These methods involve wide-field imaging, and require active control of the molecules in order to either turn emitters on or turn emitters off in order to maintain the concentration of emitters low enough to digitize the point-spread functions of individual molecules. By many imaging, photobleaching, and reactivation cycles, a superresolution image is obtained, but only for a two-dimensional projection of the actual three-dimensional sample. These methods may be collectively termed Single-Molecule Active Control Microscopy (SMACM), and have previously been applied primarily to fixed cells. However, many samples of biomedical interest, such as cells, are thick enough that two-dimensional imaging is a severe limitation. The primary goal of this research program is to achieve three-dimensional superresolution imaging in living cells using SMACM. This research will attack the problem of 3-D superresolution imaging with three thrusts. First, the optical illumination used to achieve active control will be tailored in its intensity as a function of time, in order to increase the efficiency of the reactivation and imaging process and eventually enable observation of time- dependent changes. Second, the microscope will be redesigned to utilize rotating point-spread functions. This relies on forcing the image of a single emitter to have a shape at the detector which rotates for different z- positions of the single molecule in the sample. The effect of this will be to enable much more precise determinations of the z positions of various single-molecule labels in the sample, which, when combined with precise localization in the x-y plane, will yield three-dimensional image information beyond the diffraction limit. Third, the research will implement multi-plane imaging in addition to rotating point-spread-functions, which will enable acquisition of 3D information over a greater depth into the sample. The results of this research will be to enable a new type of optical microscopy of cells, where three- dimensional superresolution information can be obtained in a noninvasive fashion about cellular substructures, including single molecules. The power of a single fluorophore as a nanoscale light source will then be used to its maximum benefit. By providing a new method for three-dimensional high resolution optical imaging in living cells, this research will bear directly upon biotechnological and biomedical applications as these fields currently utilize optical fluorescence microscopy of cells in many diagnostic situations. Current trends are pushing toward smaller and smaller spatial scales for analysis of the behavior and morphology of individual cellular structures. The ability to specifically and noninvasively analyze mutant or toxic behaviors of organelles and other tiny cellular structures will allow precise assessment of the utility of targeted drug treatments, which will help drive the future of medical interventions exactly at the point of disease.