Coherent Super-Resolution Optical Microscopy for Enhanced Image Resolution and Speed

这一建议的目的是将超分辨率荧光显微镜的分辨率和图像采集速度提高约一个数量级(到5-10 nm)。在传统的光活化定位显微镜中，分辨率受到定位精度的限制，而定位精度又与1/SQRT(N)成正比，其中N是收集到的光子的数量。在提出的方法中，我们可以获得与1/N成正比的定位精度。这是通过：(1)由于新的光栅系统而减小点扩散函数(PSF)的有效尺寸，(2)由于与高强度参考波的干涉而增加了被检测到的光子的数量，以及(3)除了使用用于光学过滤的新型光学相关器来利用信号的强度之外，还利用了位相信息。智能上的优点来自于潜在地获取大分子(亚5 nm)分辨率的荧光图像，这是通过对传统共焦显微镜的相对简单的补充来实现的。该技术的变革性是由于更高的分辨率和更快的成像速度，这将以前所未有的空间和时间分辨率阐明蛋白质和亚细胞纳米结构之间的基本相互作用。这一项目的更广泛影响包括：(1)通过创新的跨学科课程培训科学工作者；(2)通过将在3个大型外展活动中使用的实践示范模块招募代表不足的学生；(3)通过主题技术的商业化广泛传播。

The objective of this proposal is to increase the resolving power and the image acquisition speed, of super-resolution fluorescence microscopy by about an order of magnitude (to 5-10nm). Resolution in conventional photoactivatable-localization microscopy is limited by the localization precision, which, in turn is proportional to 1/sqrt(N), where N is the number of collected photons. In the proposed approach, we can achieve localization precision that is proportional to 1/N. This is achieved by: (1) reduction in the effective size of the point-spread function (PSF) as a result of a novel grating system, (2) increase in the number of detected photons due to interference with a high intensity reference wave and (3) utilization of phase information in addition to the intensity of the signals using a novel optical correlator for optical filtering.The intellectual merit arises from the potential acquisition of fluorescence images with macro-molecular (sub-5nm) resolution enabled by a relatively simple addition to conventional confocal microscopes. The transformative nature of the proposed technique is a result of the higher resolution and faster imaging, which will elucidate the fundamental interactions between proteins and sub-cellular nanostructures with unprecedented spatial and temporal resolution. Such mechanistic understanding will prove to be essential for future advancements in biology.The broader impacts of this project include (1) training of the scientific workforce via an innovative, inter-disciplinary course, (2) recruitment of under-represented students via a hands-on demonstration module to be utilized at 3 large outreach events, and (3) widespread dissemination via commercialization of the subject technology.