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

描述(申请人提供)：具有单分子主动控制的活细胞中的3D超分辨率成像单分子显微成像技术的最新进展导致了超分辨率信息的产生，即以超过标准衍射极限的分辨率观察物体的能力。这些方法涉及广场成像，需要对分子进行主动控制，以便打开或关闭发射器，以便将发射器的浓度保持在足够低的水平，以数字化单个分子的点扩散函数。通过多次成像、光漂白和再激活循环，获得了超分辨率图像，但仅针对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镜(SMACM)，以前主要应用于固定细胞。然而，许多具有生物医学意义的样本，如细胞，都足够厚，因此二维成像是一个严重的限制。这项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。这项研究将解决三推力三维超分辨率成像问题。首先，用于实现主动控制的光学照明将根据时间的函数来调整其强度，以提高重新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，以使用旋转点扩散函数。这依赖于迫使单个发射器的图像在探测器处具有形状，该形状随着样品中单个分子的不同z位置而旋转。这样做的效果是能够更精确地确定样品中各种单分子标记的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，除了旋转点扩散功能外，研究还将实现多平面成像，这将使在更深层次上获取样本的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的三维超分辨率信息，包括单分子。然后，单个荧光团作为纳米级光源的能力将被最大限度地利用。通过提供一种在活细胞中进行三维高分辨率光学成像的新方法，这项研究将直接影响到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。目前的趋势是推动空间尺度越来越小，以分析单个细胞结构的行为和形态。对细胞器和其他微小细胞结构的突变或毒性行为进行特定和非侵入性分析的能力将使人们能够准确评估靶向药物治疗的效用，这将有助于推动未来准确地在疾病发生时进行医疗干预。

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.