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

描述（由申请人提供）：单分子主动控制活细胞的三维超分辨率成像最近单分子显微成像技术的进展导致了超分辨率信息，即能够以超过标准衍射极限的分辨率观察物体。这些方法涉及宽场成像，并且需要主动控制分子，以便打开或关闭发射器，以保持发射器的浓度足够低，以数字化单个分子的点扩展函数。通过多次成像，光漂白和再激活循环，获得超分辨率图像，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活性控制显微镜（SMACM），以前主要应用于固定细胞。然而，许多生物医学兴趣样本，如细胞，足够厚，二维成像是一个严重的限制。本研究计划的主要目标是利用SMACM在活细胞中实现三维超分辨率成像。本研究将解决三维超分辨三推力成像问题。首先，用于实现主动控制的光学照明将根据时间的变化来调整其强度，以提高再激活和成像过程的效率，并最终能够观察到与时间相关的变化。其次，显微镜将被重新设计，以利用旋转点扩展功能。这依赖于迫使单个发射器的图像在探测器处具有一个形状，该形状在样品中单个分子的不同z-位置旋转。这样做的效果是能够更精确地确定样品中各种单分子标签的z位置，当与x-y平面上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，除了旋转点扩展函数外，该研究还将实现多平面成像，这将使获取更深入样品的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，可以以一种无创的方式获得关于细胞亚结构（包括单个分子）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得到最大限度的利用。通过提供活细胞三维高分辨率光学成像的新方法，该研究将直接影响生物技术和生物医学的应用，因为这些领域目前在许多诊断情况下使用细胞的光学荧光显微镜。目前的趋势是朝着越来越小的空间尺度来分析单个细胞结构的行为和形态。特异性和非侵入性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，将允许对靶向药物治疗的效用进行精确评估，这将有助于推动未来精确地在疾病点进行医疗干预。

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.