EAGER: Single Quantum Dots via 2-Photon Excitation

点击翻译按钮获取中文摘要

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.