Super-Resolution Imaging with Difference Deconvolution Microscopy

描述(申请人提供)：由于衍射限制，荧光显微镜的空间分辨率较低(最好的共聚焦显微镜为~200 nm)，但提供了关于活细胞的精细动态信息。在过去的十年里，随着新型荧光传感器和分子的发展，荧光显微镜(特别是共聚焦显微镜)在活细胞研究中的应用大大增加。我们项目的目标是使用一种新的显微镜技术，将共焦成像技术的空间分辨率提高到几十纳米。此外，我们计划在传统扫描共焦显微镜的基础上，提高DDM的时间分辨率，以便我们可以比目前使用标准扫描共焦显微镜更快地获取动态信息。我们相信，DDM一旦成功开发，将极大地提高荧光显微镜的分辨率和能力，特别是在活细胞成像中，激光功率需要足够低以避免细胞损伤，并且时间分辨率必须足够快，以解析感兴趣的动力学。该项目的目标是使用一种新的显微镜方法，将共焦成像技术的分辨率提高到几十纳米级，并以亚毫秒的时间分辨率进行活细胞成像，我们将其称为差分反卷积显微镜(DDM)。DDM使用与共聚焦显微镜类似的实验参数(例如相似的激光功率)，因此本质上适合用于脆弱的样品，如活的哺乳动物细胞。

DESCRIPTION (provided by applicant): Fluorescence microscopy has poor spatial resolution (~200nm for the best confocal microscope) owing to the diffraction limit, but provides exquisite dynamical information on live cells. With the development of novel fluorescent sensors and molecules in the past decade, the usage of fluorescence microscopy (especially confocal microscopy) for live-cell studies has greatly increased. The goal of our project is to increase the resolution of confocal imaging techniques to a few tens of nanometers in spatial resolution, using a new microscopy technique that we termed "Difference Deconvolution Microscopy (DDM)" Furthermore, we plan to increase the temporal resolution of DDM beyond that of traditional scanning confocal microscopy, so we can access even faster dynamical information than currently possible using a standard scanning confocal microscope. We believe DDM, when successfully developed, will increase dramatically the resolution and capability of fluorescence microscopy, particularly for applications in live- cell imaging where laser power needs to be sufficiently low to avoid cellular damage and the time resolution must be sufficiently fast to resolve the dynamics of interest. The goal of this project is to increase the resolution of confocal imaging techniques to the tens of nanometers in length scale and with sub-millisecond time resolution for live-cell imaging, using a new microscopy method that we termed "Difference Deconvolution Microscopy (DDM)". DDM uses comparable experimental parameters (e.g. similar laser powers) as confocal microscopy, and thus is inherently suited to use with fragile samples, such as live mammalian cells.