The objective of this program is to develop a new concept of a high throughput optical microscopy technique with unprecedented imaging resolution as well as speed. By combining two emerging fields, surface plasmon interference engineering with the structured illumination microscopy technique, the proposed plasmonic structured illumination microscopy (PSIM) takes advantage of the superior properties of surface plasmons to significantly improve the spatial resolution. A prototype plasmonic structured illumination microscope will be constructed with 3-5 fold spatial resolution improvement compared with a conventional light microscope. The target imaging speed is 50 frames/second and beyond. Intellectual Merit: The PSIM is a novel concept and it is the first ever to utilize surface plasmon interference patterns in structured illumination microscopy to improve the imaging resolution. Considering both the high resolution and the high speed, the proposed PSIM will represent a new standard of optical imaging tools that is difficult to realize through any other current techniques. Broader Impacts: The remarkable performance improvement provided by this proposed PSIM will make profound influences in a broad spectrum of fields wherever a high speed high-resolution optical microscopy is needed. The impact of this research will be far-reaching. The outreach activities include the involvement of graduate and undergraduate students in the project as well as the development of new courses which will be integrated with current under/graduate curricula. More importantly, the PSIM will also be introduced to local biologists through NCMIR, a national public imaging facility, to assist in the new discoveries in their fields.
Optical microscopes are essential tools in biological researches because light is a non-invasive probe for biological samples. However, the resolving power of conventional optical microscopy is on the order of several hundred nanometers in the visible spectrum, limited by diffraction. As more and more biological studies require sub-diffraction limited resolution to examine the structure and dynamics of the organelles, super-resolution microscopy has drawn significant attentions recently. Various advanced techniques, such as stimulated emission depletion microscopy (STED), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) etc., have been proposed and demonstrated to surpass the diffraction limit. The main goal of this research program is to develop a new super-resolution technique, termed plasmonic structured illumination microscopy (PSIM), to overcome the resolution limit of SIM by implementing the emerging plasmonic techniques. The physical principle of PSIM is similar to that of SIM. Through utilizing the Moiré fringes generated by the non-uniform illumination, SIM improves the resolution of the fluorescence microscopy by a factor of 2. The resolution enhancement factor for SIM is determined by the period of the illumination pattern, which is also limited by diffraction for the laser interference illumination used in SIM. We proposed and experimentally demonstrated that this resolution enhancement factor can be further increased by changing the illumination from the conventional laser interference to the surface plasmon interference (SPI) with sub-diffraction limited period. As a result of this program, an automated prototype multi-color PSIM system was designed and built. The system consists of three major components, the light source module, the illumination angle control module and the fluorescence image detection module. Large area nano-patterned plasmonic structures were designed and fabricated, serving as the substrates for the objects to be examined. Numerical reconstruction codes were developed to extract super-resolution information of an object from the recorded multiple diffraction limited images. In the proof-of-concept experiment, a 2.7-fold resolution improvement was achieved compared with conventional epi-fluorescence microscopy. Through further plasmonic material engineering to increase the supported surface plasmon (SP) wave vector, the PSIM could achieve even higher resolving power. Besides the proof-of-concept demonstration, various experiments were also conducted toward the application of this technique in biological studies. A low cost large-area patterned substrate fabrication technique was developed, with a thin protection layer deposited on top of the plasmonic structures to isolate the plasmonic material from the biological samples. The biocompatibility of the protected plasmonic substrate was tested by successfully growing of HeLa cells on it. Potentially, the demonstrated wide field PSIM technique could be used for high-speed, super-resolution biomedical studies. Moreover, the developed prototype PSIM system could be used for other plasmonic assisted super-resolution techniques as well. This research program provided comprehensive trainings for two Ph. D students in the field of nanofabrication and optical microscopy. They gained extensive experiences in clean-room fabrication, optical system design, alignment and image reconstruction. This project resulted the publication of multiple papers on peer-reviewed research journals and several oral presentations at international conferences in the plasmonics field. Moreover, this project is a major part of the thesis of one Ph. D student, and she successfully graduated at the end of this program.
