Super-Resolution Imaging with Difference Deconvolution Microscopy

描述（由申请人提供）：由于衍射极限，荧光显微镜的空间分辨率较差（最好的共聚焦显微镜约为200nm），但可以提供活细胞的精细动态信息。近十年来，随着新型荧光传感器和荧光分子的发展，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。我们项目的目标是将共聚焦成像技术的分辨率提高到几十纳米的空间分辨率，使用一种新的显微镜技术，我们称之为“差分反卷积显微镜（DDM）”。此外，我们计划将DDM的时间分辨率提高到传统扫描共聚焦显微镜之外，这样我们就可以比目前使用标准扫描共聚焦显微镜更快地获取动态信息。我们相信，一旦成功开发，DDM将显著提高荧光显微镜的分辨率和能力，特别是在活细胞成像应用中，激光功率需要足够低以避免细胞损伤，时间分辨率必须足够快以解决感兴趣的动态。该项目的目标是使用一种新的显微镜方法，我们称之为“差分反褶积显微镜（DDM）”，将共聚焦成像技术的分辨率提高到几十纳米的长度尺度和亚毫秒的活细胞成像时间分辨率。DDM使用与共聚焦显微镜类似的实验参数（例如，类似的激光功率），因此本质上适合用于易碎样品，例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.