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.
