EAGER: Single Quantum Dots via 2-Photon Excitation

《通过双光子激发的单量子点》，保罗·R·塞文，智力价值。双光子荧光显微镜将被用来激发附着在体外和体内生物标本上的单个量子点(Q点)。最终，目标是展示超精度和超分辨率，即使用将开发的方法超过显微镜的衍射限制(10 Nm)的精度和分辨率。双光子激发已被证明是一种非常有用的手段，可以在x、y和z方向同时获得分辨率(使用称为共焦显微镜的技术)，最小化光散射的影响，并用单一激发波长同时激发多个荧光团。Q点的单光子激发已经作为一种非常有用的手段来实现非常明亮的Q点发射，长时间的光稳定性，以及在单分子水平上显示单个Q点的准确性。在这个项目中，初步结果表明，在依靠极低的光强度来激发Q点的增强条件下，也可以很容易地用双光子显微镜观察到单个Q点。该项目的一个目标是在生物学条件下优化这些双光子激发条件，以便例如观察分子马达的运动(例如，肌动蛋白和肌球蛋白)，或在感染细菌的过程中捕获噬菌体病毒。PI已经在宽场激发(即不将光聚焦到衍射限制的光斑)下获得了有希望的初步结果，但没有z分辨率所需的共焦效应。为了获得z分辨率，研究人员将重复通常的方法，但将在两个重要方面进行改进。首先，将使用高灵敏度的电荷耦合探测器(CCD)摄像头来提高图像采集的速度。其次，空间光调制器将被用来以10×10阵列的近衍射限制光点来打击样品，进一步提高速度，并在所有三个空间维度上都允许超精度和超分辨率。更广泛的影响。该项目更广泛的目标是通过开发和生物应用双光子激发单个Q点来实现新的生物发现。许多技术都非常简单(或者可能会变得非常简单)。因此，预计该项目将产生通用技术，这些技术将广泛应用于许多对显示活细胞和其他生物样本中的单分子感兴趣的实验室。该项目还将加强将参与该项目的本科生和研究生的培训和专业知识。

Lay Abstract0968976EAGER: Single Quantum Dots via 2-Photon ExcitationSelvin, Paul R. Intellectual merit. A two-photon fluorescence microscope will be used to excite individual quantum dots (q-dots), attached to in vitro and in vivo biological specimens. Eventually, the aim is to demonstrate super-accuracy and super-resolution, i.e. accuracy and resolution beyond the diffraction limit of the microscope ( 10 nm) using the methods that will be developed. Two-photon excitation has been shown to be incredibly useful as a means to obtain simultaneous resolution in x, y, and z directions (using the technique known as confocal microscopy), to minimize the effect of light scattering, and to excite multiple fluorophores simultaneously with a single excitation wavelength of light. One-photon excitation of q-dots has already been extremely useful as a means to achieve very bright q-dot emission, long photostability, and the accuracy to visualize individual q-dots at the single-molecule level. In this project, preliminary results indicate that individual q-dots can also be easily visualized with two-photon microscopy under enhanced conditions that rely on extremely low light intensities to excite the q-dots. A goal of this project is to optimize these conditions for 2-photon excitation under biological conditions in order, for instance, to watch molecular motors move (e.g., kinesin and myosin), or to catch a phage virus in the act of infecting a bacterium. The PI has obtained promising preliminary results with wide-field excitation (i.e., not focusing the light down to a diffraction-limited spot) but without a confocal effect needed for z-resolution. To obtain z-resolution, the researchers will repeat the usual methodology but will enhance it in two important ways. First, a highly sensitive charge-coupled detector (CCD) camera will be used to increase the speed of image acquisition. Second, a spatial light modulator will be used to hit the sample with a 10 × 10 array of near-diffraction limited spots, further increasing speed and also permitting super-accuracy and super-resolution in all three spatial dimensions. Broader impacts. The broader goals of the project are to enable new biological discovery through the development and biological application of two-photon excitation of individual q-dots. Many of the techniques are (or potentially will become) extremely simple. Therefore, it is anticipated that the project will result in generalized techniques that will be widely available to many laboratories interested in visualizing single molecules in living cells and other biological specimens. The project will also enhance the training and expertise of the undergraduates and graduate students who will be involved in the project.